ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B.

Members of the ICE/Ced-3 gene family are likely effector components of the cell death machinery. Here, we characterize a novel member of this family designated ICE-LAP6. By phylogenetic analysis, ICE-LAP6 is classified into the Ced-3 subfamily which includes Ced-3, Yama/CPP32/apopain, Mch2, and ICE-LAP3/Mch3/CMH-1. Interestingly, ICE-LAP6 contains an active site QACG pentapeptide, rather than the QACG pentapeptide shared by other family members. Overexpression of ICE-LAP6 induces apoptosis in MCF7 breast carcinoma cells. More importantly, ICE-LAP6 is proteolytically processed into an active cysteine protease by granzyme B, an important component of cytotoxic T cell-mediated apoptosis. Once activated, ICE-LAP6 is able to cleave the death substrate poly(ADP-ribose) polymerase into signature apoptotic fragments.

Apoptosis, or programmed cell death, is a physiologic process important in the normal development and homeostasis of metazoans (1). It is becoming apparent that a class of cysteine proteases homologous to Caenorhabditis elegans Ced-3 play the role of "executioner" in the apoptotic mechanism (2)(3)(4). In the nematode, two proteins, encoded by ced-3 and ced-4, are required for all somatic cell deaths that occur during development (5). Mutations of ced-3 and ced-4 abolish the apoptotic capability of cells that normally die during C. elegans embryogenesis (6). While no mammalian homologs of ced-4 have been identified, ced-3 shares sequence similarity with interleukin-1␤ converting enzyme (ICE) 1 (7), a cysteine protease involved in the processing and activation of pro-interleukin-1␤ to an active cytokine (8,9). Recently, numerous homologs of ICE/ Ced-3 have been characterized, comprising a new gene family of cysteine proteases.
Based on similarities with the structural prototype interleukin-1␤ converting enzyme, ICE/Ced-3 family members are synthesized as zymogens that are capable of being processed to form active heterodimeric enzymes (9). It will be important to determine which family members are in fact activated in response to apoptotic stimuli. Previous studies have demonstrated that pro-Yama and pro-ICE-LAP3 are processed into active subunits in response to various death stimuli including engagement of Fas/APO-1 or treatment with staurosporine (18,21). Further, the serine protease granzyme B, one of the major effectors of cytotoxic T cell-mediated apoptosis, was shown to directly activate Yama (but not ICE), in vitro (22,23).
Here we report the cloning and characterization of a novel member of the ICE/Ced-3 gene family designated ICE-LAP6 (for ICE-Like Apoptotic Protease 6). Based on sequence homology, ICE-LAP6 is classified in the subset of family members most related to C. elegans Ced-3 including Yama, ICE-LAP3, and Mch2. Interestingly, ICE-LAP6 contains a unique active site pentapeptide (QACGG rather than QACRG), which distinguishes it from other family members. Overexpression of ICE-LAP6 in MCF7 breast carcinoma cells induces cell death and mutation of the putative catalytic cysteine residue abolishes its apoptotic potential. Furthermore, granzyme B directly activates ICE-LAP6 and Yama in vitro, suggesting that granzyme B may mediate its cytotoxic effect via activation of several ICE/Ced-3 family members. Once activated, Yama and ICE-LAP6 are both able to cleave the DNA repair enzyme poly-(ADP-ribose) polymerase (PARP) into signature apoptotic fragments. Taken together, our results suggest that ICE-LAP6, like other members of the Ced-3 subfamily, may have an important role in the apoptotic mechanism.

Cloning of Human ICE-LAP6 -
The cDNA corresponding to the partial open reading frame of ICE-LAP6 was identified as a sequence homologous to ICE-LAP3 (18) on searching the Human Genome Sci-* This work was supported in part by National Institutes of Health Grant CA64803. 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 /EBI Data Bank with accession number(s) U567390. ences private data base using established EST methods (24,25). This clone contains an open reading frame encoding the C-terminal 300 amino acids of ICE-LAP6. Full-length cDNAs were obtained by screening an oligo(dT)-primed cDNA library of the human chronic myelogenous leukemia cell line K562 (in pCDM8 vector and kindly provided by Dr. John Lowe, University of Michigan). 1 ϫ 10 6 transformants were screened with a 32 P-labeled DNA fragment generated by PCR, corresponding to nucleotides 615 to 940 of the ICE-LAP6 open reading frame (26). Double-stranded DNA sequencing was carried out by the dideoxy chain termination method using modified T7 DNA polymerase (Sequenase, U. S. Biochemical Corp.). Sequence alignments were performed using DNASTAR Megalign software.
Northern Blot Analysis-Adult and fetal human multiple tissue Northern blots (Clontech) containing 2 g/lane poly(A) ϩ RNA were hybridized, according to the manufacturer's instructions, using the same 32 P-labeled ICE-LAP6 probe used for library screening.
Expression Vectors-The DNA inserts encoding the C-terminal FLAG-tagged (ICE-LAP6 flag) or His 6 -tagged (ICE-LAP6 His) ICE-LAP6 were generated by PCR and subcloned into the mammalian expression vector pcDNA3 (Invitrogen). The 5Ј PCR primer (GAACGG-GGTACCGCCATGGACGAAGCGGATCGGC) contained a custom KpnI restriction site (underlined) and the two 3Ј primers(TGCTCTAGATT-ACTTGTCATCGTCGTCCTTGTAGTCTGATGTTTTAAAGTTAAGT-TTTTTCCGGAG) or (TGCTCTAGATTAGTGGTGGTGGTGGTGGT-GTGATGTTTTAAAGAAAAGTTTTT TCCGGAG) encoded a FLAG epitope tag (DYKDDDDK, italics) or a His6 tag (bold), respectively. Alteration of the active site cysteine 286 to an alanine was accomplished by site-directed mutagenesis employing a four-primer PCRbased method (27). The mutagenetic oligonucleotides were AAGCT-CTTTTTCATCCAGGCCGCGGGTGGGGAGCAGAAGAC and GTCTT-TCTGCTCCCCACCCGCGGCCTGGATGAAAAAAGC. The presence of the introduced mutation (underlined) and fidelity of PCR replication were confirmed by sequence analysis.
Apoptosis Assay-MCF7 breast carcinoma cells were transiently transfected as described previously (28). Briefly, 2.5 ϫ 10 5 MCF7 cells were transfected with 0.25 g of the reporter plasmid pCMV ␤-galactosidase plus 1 g of test plasmid in 6-well tissue culture dishes using Lipofectamine as per manufacturer's instructions. The transfection was carried out in 1 ml of Opti-MEM Minimal Media (Life Technologies, Inc.) and after 5 h, 1 ml of serum-containing growth media was added. Two days later, the cells were fixed with 0.5% glutaraldehyde and stained with X-gal for 4 h. Cells were visualized by phase-contrast microscopy. At least 300 ␤-galactosidase-positive cells were counted for each transfection (n ϭ 3) and identified as apoptotic or nonapoptotic based on morphological alterations typical of adherent cells undergoing apoptosis including becoming rounded, condensed, and detaching from the dish (29).
Expression and Purification of His 6 -tagged Yama and His 6 -tagged ICE-LAP6 - 35 S-Labeled Yama and ICE-LAP6 proteins were generated by in vitro transcription/translation using the TNT kit (Promega) according to the instructions of the manufacturer; the template plasmids were ICE-LAP6 His and Yama His (15). The translated proteins were purified by chromatography as described previously (15).
Activation of ICE-LAP6 and Yama by Granzyme B-Purified in vitro translated pro-ICE-LAP6 or pro-Yama was activated by incubation with granzyme B as described previously (22). Briefly, 48 l of 35 Slabeled protein were incubated with 20 pmol of purified granzyme B (22) in a total volume of 50 l. After 4 h, 20 l of reaction was removed for SDS-PAGE analysis. 520 pmol of anti-GraB (22) was added to the rest of the reaction mix to neutralize granzyme B activity. Following a 15-min incubation, 1 l (150 ng) of purified PARP (15) was added, and the reaction was allowed to proceed for 2 h. The control reaction containing PARP alone or PARP plus granzyme B and anti-GraB was carried out under identical conditions, except that Yama or ICE-LAP6 was not added. The reaction buffer contained 50 mM Hepes (pH 7.4), 0.1 M NaCl, 0.1% CHAPS, and 10% sucrose. All incubations were carried Based on the x-ray crystal structure of ICE, the conserved residues involved in catalysis are marked with filled circles; filled triangles represent the binding pocket for the carboxylate of the P 1 Asp; filled squares indicate the residues adjacent to the P 2 -P 4 amino acids.
out at 37°C in 10 mM dithiothreitol. Samples were analyzed by immunoblotting with anti-PARP monoclonal antibody C-2-10 as described previously (15).

RESULTS AND DISCUSSION
Cloning of ICE-LAP6 -The Human Genome Sciences human cDNA data base was searched for genes related to the ICE-LAP3 peptide sequence (18). A novel cDNA clone, encoding a partial open reading frame, was identified and showed sequence homology with members of the ICE/Ced-3 gene family. To obtain a full-length cDNA, a human chronic myelogenous leukemia cell (K562) cDNA library was screened. Of 22 positive clones, 6 clones yielded a 2.3-kilobase cDNA containing an 1252-base pair open reading frame that encoded a novel protein with a predicted molecular mass of 45.8 kDa, designated ICE-LAP6 (Fig. 1A). The putative initiator methionine (GCCATGG) was in agreement with the consensus Kozak's sequence for translation initiation (30).

ICE-LAP6 Is a Novel Member of the ICE/Ced-3 Gene
Family-A BLAST search of GenBank protein data base revealed that the predicted protein sequence of ICE-LAP6 has significant similarity to the members of the ICE/Ced-3 family, particularly in the regions corresponding to the active subunits of ICE (9). In this region, ICE-LAP6 shares 31% sequence identity (55% sequence similarity) with the C. elegans Ced-3 protein, 33% identity (52% sequence similarity) with ICE-LAP3, 30% identity (56% similarity) with Mch2␣, and 29% sequence identity (52% similarity) with Yama. ICE-LAP6 also has 25-28% sequence identity with ICE and the ICE-related genes, ICE rel II and ICE rel III. Phylogenetic analysis of the ICE/Ced-3 gene family showed that ICE-LAP6 is a member of the Ced-3 subfamily which includes Yama, ICE-LAP3, and Mch2 (Fig. 1B). Like Ced-3, ICE-LAP6 contains a long N-terminal putative prodomain.
Based on the x-ray crystal structure of ICE (31,32), the amino acid residues His 237 , Gly 238 , and Cys 285 of ICE are involved in catalysis, while the residues Arg 179 , Gln 283 , and Arg 341 form a binding pocket for the carboxylate side chain of the P 1 aspartic acid. These six residues are conserved in all ICE/Ced-3 family members thus far cloned as well as in ICE-LAP6. However, residues that form the P 2 -P 4 binding pockets are not widely conserved among family members, suggesting that they may determine substrate specificity. Interestingly, ICE-LAP6 contains a unique active site pentapeptide QAC-GG, instead of the QACRG shared by other family members (Fig. 1C).
Distribution of ICE-LAP6 -Northern blot analysis revealed that ICE-LAP6 is constitutively expressed in a variety of fetal and adult human tissues (Fig. 2). Two ICE-LAP6 mRNA transcripts were detected (Fig. 2). The 2.3-kilobase transcript corresponds to the size of the cDNA clones isolated from the K562 library. The other transcript, which is approximately 3 kilo-bases, may represent an alternatively spliced ICE-LAP6 isoform.
Overexpression of ICE-LAP6 in MCF7 Cells Induces Apoptosis-To study the functional role of ICE-LAP6, we transiently transfected MCF7 breast carcinoma cells with an expression vector encoding the full-length ICE-LAP6 protein (ICE-LAP6flag) and subsequently assessed for apoptotic features. Like the other ICE/Ced-3 family members, expression of ICE-LAP6 caused cell death (Fig. 3A). The ICE-LAP6-transfected MCF7 cells displayed morphological alterations typical of adherent cells undergoing apoptosis, becoming rounded, condensed, and detaching from the dish (Fig. 3B). ICE-LAP6 induced apoptosis was inhibited by the broad spectrum ICE inhibitor z-VAD fmk (33) (data not shown). To determine whether the amino acid residue Cys 286 , corresponding to the catalytic Cys 285 of ICE, was essential for apoptotic activity, a mutant form of ICE-LAP6 was generated in which the cysteine residue was altered to an alanine. As predicted, overexpression of the mutant form of ICE-LAP6 did not induce apoptotic changes in MCF7 cells (Fig. 3, A and B). Furthermore, these results demonstrate that an ICE/Ced-3 family member containing an active site QACGG pentapeptide (rather than QACRG) may still possess apoptosis-inducing potential and presumably enzymatic activity.
Proteolytic Activation of ICE-LAP6 by Granzyme B-Members of the ICE/Ced-3 gene family are synthesized as proenzymes and activated by proteolytic cleavage at specific aspartate residues to form heterodimeric enzymes. In ICE, this cleavage removes the prodomain and produces a heterodimeric complex consisting of p20 and p10 subunits (9). Similarly, activated Yama is comprised of two subunits, p17 and p12, which are derived from a 32-kDa proenzyme (17). The mechanism by which death signals activate ICE/Ced-3 family members is poorly understood. Recent studies on granzyme B, however, suggest that cytotoxic T cells may utilize this secreted serine protease to directly activate members of the ICE/Ced-3 family. It has been demonstrated that granzyme B can proteolytically activate pro-Yama, generating an active enzyme capable of cleaving the death substrate PARP into characteristic fragments (22,23). By contrast, ICE, although cleaved by granzyme B, fails to be activated (22).
Thus, we determined whether ICE-LAP6 can serve as a substrate for granzyme B. His 6 tagged ICE-LAP6 and Yama were generated by in vitro transcription/translation, and subsequently purified by Ni-affinity chromatography as described under "Materials and Methods." The purified in vitro translated pro-ICE-LAP6 or pro-Yama was incubated with purified granzyme B (34,35). After 4 h at 37°C, ICE-LAP6 was proteolytically processed into three fragments. The two low molecular weight bands represent the active subunits of ICE-LAP6 and correspond to the p17 and p12 subunits of active Yama (Fig.  4A). The doublet at 17 kDa is likely generated by differential cleavage following either of the two aspartic acid residues that are distal to the QACGG motif and are circled in Fig. 1A. The 32-kDa band is an likely intermediate, in which only the prodomain is removed (a similar intermediate is generated in the activation of ICE-LAP3) (18,21). Next, we assessed whether granzyme B-mediated cleavage of ICE-LAP6 generates an active enzyme by assaying for PARP cleavage. PARP is proteolyzed during many forms of apoptosis, and the enzyme(s) responsible is likely of the ICE/Ced-3 family. To exclude the possibility of direct cleavage of PARP by granzyme B, granzyme B-processed ICE-LAP6 and Yama were incubated with a selective inhibitor of granzyme B (anti-GraB) as described previously (22). Interestingly, both granzyme B-processed Yama and ICE-LAP6 were active as determined by their ability to cleave PARP (Fig. 4B). Unlike ICE, ICE-LAP6 and other members of the Ced-3 subfamily are able to cleave the PARP into signature apoptotic fragments (15-17, 19, 20, 36).
In conclusion, we have identified a novel member of the ICE/Ced-3 family of cysteine proteases. ICE-LAP6 has a unique active site QACGG pentapeptide and is classified in the subfamily most related to Ced-3 and Yama. Ectopic expression of ICE-LAP6 in mammalian cells causes apoptosis. Importantly, ICE-LAP6, like Yama, was directly activated by granzyme B in vitro, suggesting that cytotoxic T cells may mediate apoptosis by activating more than one ICE/Ced-3 family member in susceptible target cells. Yama, ICE-LAP3, and now ICE-LAP6, have been shown to be proteolytically activated by apoptotic stimuli. The cloning and characterization of ICE-LAP6 will enhance our understanding of the cell death machinery and the proteases that compose it. Additionally, it will necessary to develop specific inhibitors for each of the three functionally indistinguishable ICEs (ICE-LAP6, ICE-LAP3 and Yama) or inactivation of their genes by homologous recombination before one can discern the contribution of each to granzyme B mediated apoptosis. and is presumed to correspond to a form of ICE-LAP6 in which the prodomain is removed. p17 and p12 indicate the active subunits of Yama and ICE-LAP6. B, granzyme B-processed ICE-LAP6 cleaves PARP to an 85-kDa apoptotic fragment. After incubation with granzyme B (GraB) as described above, anti-GraB was added to the rest of the reaction mix to neutralize granzyme B activity. Following a 15-min incubation, purified PARP was added, and the reaction was allowed to proceed for 2 h. Samples were analyzed by SDS-PAGE and subsequent immunoblotting with anti-PARP monoclonal antibody C-2-10.