©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Processing and Activation of CMH-1 by Granzyme B (*)

(Received for publication, January 31, 1996; and in revised form, March 12, 1996)

Yong Gu Charlyn Sarnecki Mark A. Fleming Judith A. Lippke R. Chris Bleackley (1) Michael S.-S. Su (§)

From the From Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139 Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Granzyme B plays an essential role in cytotoxic T lymphocyte (CTL)-mediated cell killing. Recent studies suggest that granzyme B may exert its effect by cleaving and activating CPP32, a member of the interleukin-1beta-converting enzyme/Ced-3 family of cysteine proteases. We have examined the processing and activation of CMH-1, a close homologue of CPP32, by granzyme B in vitro. We have found that granzyme B specifically cleaves CMH-1 at Asp-Ser between the p20 and p12 and activates the cysteine protease. Cleavage between p20 and the prosequence of CMH-1 at Asp-Ala is autocatalytic and is not required for CMH-1 activity in vitro. The cleavage and activation of CMH-1 by granzyme B in vitro suggest that, in addition to CPP32, CMH-1 may also play a role in CTL-mediated cell killing.


INTRODUCTION

Cytotoxic T lymphocytes (CTLs) (^1)contribute to host cell-mediated immunity against viral pathogens, parasites, and neoplastic transformation as well as to host pathology associated with transplant rejection and autoimmune diseases (for review see (1) ). CTLs destroy target cells in part by secreting perforin along with a family of serine proteases, the granzymes, into the intercellular space between the effector and target cell. Perforin, a pore-forming protein, facilitates the entry of the granzymes into target cells where they trigger apoptosis by an unknown mechanism(2) . One of the granzymes, granzyme B, is a serine protease with a substrate specificity for aspartate at P1 and is essential for the rapid induction of DNA fragmentation and apoptosis in target cells(3) .

Recently, Darmon et al.(4) reported that granzyme B can cleave and activate CPP32, a member of the interleukin-1beta-converting enzyme (ICE)/Ced-3 family of cysteine proteases that also has a substrate specificity for Asp-X. CPP32 has been isolated based on its ability to cleave poly(ADP-ribose) polymerase (PARP)(5) , a DNA repair enzyme that is rapidly proteolyzed in apoptotic cells(6) . It has become more apparent that the ICE/Ced-3 family of proteases plays a critical role in mediating apoptosis (see (7) for review). Genetic studies in the nematode Caenorhabditis elegans indicate that the ced-3 gene is indispensable for cell death during development(8) . Inhibitors of ICE-like proteases can prevent mammalian cell death induced by a variety of factors(5, 9, 10) . Furthermore, thymocytes derived from ICE-deficient mice are resistant to Fas-induced cell death, suggesting a physiological role for ICE in Fas-mediated apoptosis(11) . Thus the regulation of these proteases may be critical in executing apoptotic cell death.

Each of the ICE/Ced-3 family of proteases is synthesized as an inactive precursor polypeptide requiring proteolytic cleavage at specific aspartate residues to produce two subunits of approximately 20 kDa (p20) and 10 kDa (p10), which together form the active protease (see (12) ). The precursor proteins contain N-terminal prosequences of various lengths followed by the p20 and p10 subunits. In some members of the protease family, such as ICE, a short linker sequence exists between the p20 and p10 subunits. Processing and activation of these proteases in vivo remain unclear. Some precursors seem to undergo autocatalytic cleavage; others may require distinct aspartate-specific proteases for activation. Therefore, identification of CPP32 as a substrate for granzyme B suggests CTL-mediated cell death may be initiated by a protease cascade involving the ICE/Ced-3 family of proteases.

Recently, a new member of the ICE/Ced-3 family of proteases, CMH-1 (also named MCH-3 and ICE-LAP3), has been identified(13, 14, 15) . It has the highest homology to CPP32 and is able to cleave PARP and induce apoptosis in COS cells. In this paper, we examined the processing and activation of CMH-1 by granzyme B in vitro. We report that granzyme B cleaves CMH-1 specifically between p20 and p12 and activates the CMH-1 protease. The removal of the prosequence is an autocatalytic event and is not required for CMH-1 activity in vitro. The cleavage and activation of CMH-1 by granzyme B in vitro suggests that, in addition to CPP32, CMH-1 may also play a role in CTL-mediated cell killing.


MATERIALS AND METHODS

Expression and Purification of Active Murine Granzyme B in COS Cells

cDNA encoding the murine granzyme B lacking the activation dipeptide was described previously(16) . Polymerase chain reaction was used to add a (His)(6) tag to the C terminus. For COS cell expression, amplified cDNA was cloned into the mammalian expression vector pCDLSRalpha(17) . COS cell transfections were performed as described(18) . For purification of granzyme B, five 15-cm dishes (3.6 times 10^6 cells/dish) of COS cells were transfected and lysed in 1 ml of lysis buffer (50 mM Tris, pH 8.0, 390 mM NaCl, 1% Triton X-100). Lysates were cleared by centrifugation at 100,000 times g for 30 min, and the supernatant (8.5 mg of protein) was loaded onto an SP-Sepharose (Pharmacia Biotech Inc.) column, washed with lysis buffer containing 400 mM NaCl, and then eluted with 1 ml of lysis buffer containing 500 and 700 mM NaCl. Eluted fractions were pooled and incubated with a small amount of nickel-agarose beads (Qiagen) for 1 h at 4 °C. After washing the beads with lysis buffer containing 600 mM NaCl and 25 mM imidazole, bound proteins were eluted with 0.5 ml of 200 mM imidazole in the same buffer. SDS-PAGE and silver staining indicated that the eluate contained two major polypeptides of 31 and 34 kDa at approximately 1-2 µg/ml. Using the standard granzyme B substrate Boc-Ala-Ala-Asp-thiobenzyl (where Boc is t-butyloxycarbonyl) ester, we determined the activity of the preparation to be 100 units/ml, where 1 unit was defined as the amount of enzyme required to hydrolyze 1 nmol of substrate/min(19) , giving rise to an estimated specific activity of 50,000-100,000 units/mg. Unless otherwise noted, this preparation was used as the purified granzyme B for all experiments described in this report.

Expression and Purification of Recombinant (His)(6)-tagged CMH-1 and CPP32 Proteins in Escherichia coli

cDNA encoding the full-length CMH-1 was described previously(13) . cDNAs encoding mutant CMH-1 proteins were generated by site-directed mutagenesis (20) and confirmed by DNA sequencing. Expression plasmids for N-terminally (His)(6)-tagged wild type or mutant CMH-1 were constructed by introducing XhoI sites at the 5` and 3` ends of CMH-1 cDNAs by polymerase chain reaction using primers 5`CGGCTGCAGCTCGAGGCAGATGATCAGGGCTGTATTGAG and 5` GGGCTCGAGCTATTGACTGAAGTAGAGTTC and then ligating the resulting XhoI fragments into a XhoI-cut E. coli expression vector pET-15b (Novagen). The resulting plasmids direct the synthesis of polypeptides of 325 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 at Ala^2, as confirmed by DNA sequencing and by N-terminal sequencing of the expressed proteins. E. coli strain BL21(DE3) carrying a particular expression plasmid was induced with 0.8 mM isopropyl-1-thio-beta-D-galactopyranoside for 2 h at 30 °C, harvested, and lysed in a Microfluidizor (Microfluidic, Watertown, MA) in Buffer A (20 mM sodium phosphate, pH 7.4, 300 mM NaCl, 2 mM dithiothreitol, 10% glycerol, 0.4 mM phenylmethylsulfonyl fluoride, and 2.5 µg/ml leupeptin). Lysates were cleared by centrifugation at 100,000 times g for 30 min, and the supernatant (S100) was loaded onto a nickel-agarose column. After washing with Buffer A containing 25 mM imidazole, CMH-1 protein was eluted with 50-100 mM imidazole in Buffer A. The eluate was desalted and applied onto a DEAE-Sepharose column in 20 mM Tris-HCl (8.0), washed with the same buffer containing 50 mM NaCl, and eluted with the same buffer containing 100 mM NaCl. N-terminally (His)(6)-tagged full-length CPP32 active site cysteine mutant (C163S) protein was expressed and purified similarly, except that the nickel-agarose chromatography was run at pH 8.0 and DEAE-Sepharose chromatography at pH 8.4.

Cleavage of CPP32 and CMH-1 by Granzyme B in Vitro

S-Labeled CPP32 and CMH-1 were prepared by in vitro transcription and translation using the TNT T7-coupled reticulocyte lysate system (Promega) and [S]methionine (Amersham Corp.). Labeled proteins were incubated for 30-60 min at 37 °C either with crude lysates of granzyme B-expressing COS cells or with purified granzyme B in a total volume of 30 µl in a buffer containing 20 mM Hepes (pH 7.0), 60 mM NaCl, and 0.2 mM EDTA. Cleavage products were analyzed by SDS-PAGE and fluorography. For cleavage of purified CPP32 or CMH-1 by granzyme B, purified (His)(6)-CMH-1 or (His)(6)-CPP32 proteins were used in place of the in vitro translated proteins. Cleavage products were analyzed by SDS-PAGE and Coomassie Blue staining.

Other Methods

Cleavage of S-labeled PARP by CMH-1 in vitro was carried out as described(13, 21) . Expression plasmids were confirmed by sequencing the entire coding sequence using the ABI 373A DNA sequencer, and N-terminal amino acid sequencing was performed using the ABI 477A protein sequencer.


RESULTS AND DISCUSSION

CMH-1 Is Cleaved More Efficiently than CPP32 by Granzyme B

To determine if CMH-1 can be cleaved by granzyme B, we incubated CMH-1 produced by in vitro translation with a crude lysate prepared from COS cells expressing the active form of murine granzyme B from an expression vector. We found that CMH-1 is cleaved from a 35-kDa polypeptide into two peptides of 23 and 12 kDa upon incubation with lysates prepared from transfected cells. Incubation of CMH-1 with lysates from mock transfected cells resulted in no cleavage of the 35-kDa peptide (Fig. 1A). This cleavage was not affected by the presence of tetrapeptide aldehydes Ac-DEVD-CHO or Ac-YVAD-CHO (data not shown), potent inhibitors of CPP32, CMH-1, and ICE(5, 13, 22) , indicating that the cleavage of CMH-1 was not mediated by an ICE- or CPP32-like protease(s) and that granzyme B is not sensitive to these two inhibitors.


Figure 1: CMH-1 is cleaved more efficiently than CPP32 by granzyme B in vitro. A, in vitro translated S-labeled CMH-1 (lanes 1-3) or CPP32 (lanes 4-6) was incubated for 30 min with buffer (lanes 1 and 4) or with 4 µl of crude lysates (35 µg of protein) from mock transfected (lanes 2 and 5) or granzyme B-transfected COS cells (lanes 3 and 6) as described under ``Materials and Methods.'' Cleavage products were analyzed by SDS-PAGE (16%) and fluorography. Numbers on the left represent molecular mass in kilodaltons. Positions of the uncleaved CMH-1 (35 kDa) and CPP32 (33 kDa) are indicated on the right along with their respective cleavage products (CMH-1, 23 and 12 kDa; and CPP32, 21 and 12 kDa). B, 2 µg of purified (His)(6)-tagged CMH-1-C186S (lanes 1-6) and CPP32-C163S (lanes 7 and 8) were incubated with the indicated amounts of the purified granzyme B (GranB) for 1 h at 37 °C and analyzed by SDS-PAGE and Coomassie Blue staining. Lane M, molecular mass markers in kilodaltons. Positions of uncleaved CMH-1 and CPP32 were indicated along with their cleavage products (open arrowheads).



To compare the ability of granzyme B to cleave CPP32, we incubated CPP32 produced by in vitro translation with the COS cell lysates and found that the 33-kDa CPP32 was cleaved into 21- and 12-kDa peptides, as observed by Darmon et al.(4) . Interestingly, we found that CMH-1 appeared to be cleaved more efficiently than CPP32 by granzyme B (Fig. 1A). To confirm this result, we purified granzyme B from granzyme B-expressing COS cells and then incubated equal amounts (2 µg) of purified (His)(6)-tagged CPP32-C163S or CMH-1-C186S proteins (see below and ``Materials and Methods'') with various amounts of the purified enzyme. We found that CPP32 cleavage required a 25-50-fold higher concentration of granzyme B than CMH-1 cleavage (Fig. 1B). Therefore, under our experimental conditions, CMH-1 is a superior substrate for granzyme B than CPP32.

Cleavage of CMH-1 by Granzyme B Leads to Activation of the Cysteine Protease

In order to determine if the cleavage of CMH-1 by granzyme B would lead to its activation, we expressed an N-terminally (His)(6)-tagged full-length CMH-1 in E. coli and purified the protein by nickel-affinity chromatography followed by DEAE anion-exchange chromatography (Fig. 2). The purified CMH-1 protein contained approximately equal amounts of two major polypeptides of 37 kDa (p37) and 32 kDa (p32). N-terminal amino acid sequencing and immunoblotting with anti-(His)(6) antibody (Dianova) indicated that p37 contains the (His)(6) tag fused to CMH-1 at position Ala^2 as expected for the full-length protein while p32 starts at Ala, which is preceded by an aspartate (Asp). The relative amount of p32 tends to increase during purification and storage, suggesting that p32 is derived from p37 by autocleavage at Asp. A small amount of 20- and 12-kDa peptides was also visible. This is probably due to an autocatalytic cleavage of p32 between p20 and p12 (see below) although it is clearly a slow and inefficient process. Rechromatography of the DEAE fraction on the nickel-agarose column resulted in a co-purification of p32 and p37, indicating that p32 is associated with p37 and that the CMH-1 precursor protein exists as dimers or higher order oligomers (Fig. 2)(18) .


Figure 2: Purification of (His)(6)-tagged CMH-1 from E. coli. (His)(6)-CMH-1 was expressed and purified as described under ``Materials and Methods,'' analyzed by SDS-PAGE on a 16% gel, and visualized by Coomassie Blue staining. Lane 1, molecular mass marker; lane 2, crude cell lysates (140 µg); lane 3, nickel-agarose column flow-through (140 µg); lane 4, nickel-agarose eluate (7 µg); and lane 5, DEAE-Sepharose eluate (5 µg). Lane 6, DEAE-Sepharose eluates rechromatographed on nickel-agarose column (4 µg).



Purified CMH-1 (2 nM) had no detectable protease activity against PARP produced by in vitro translation (Fig. 3, B and C), a known substrate for mature CMH-1(13, 14) . To determine if cleavage of CMH-1 by granzyme B would lead to its activation, CMH-1 was treated with purified granzyme B (Fig. 3B) and then incubated with the PARP substrate. We found that PARP cleavage was readily detected at nanomolar CMH-1 concentrations and was inhibited by Ac-DEVD-CHO (Fig. 3C). Since granzyme B itself had no detectable activity against PARP (Fig. 3C), these results indicate that cleavage of CMH-1 by granzyme B leads to the activation of the CMH-1 protease.


Figure 3: Processing and activation of CMH-1 by granzyme B. A, schematic drawing of (His)(6)-CMH-1 with processing sites (D23-A24 and D198-S199) and resulting fragments indicated. B, purified CMH-1 (2 µg) was incubated for 30 min at 37 °C with 0.3 unit of purified granzyme B or buffer in the presence or absence of 8 µM Ac-DEVD-CHO as indicated and analyzed by SDS-PAGE. Proteins were visualized by Coomassie Blue staining (lanes 1-6) or by immunoblotting with an anti-(His)(6) antibody (lanes 7-10). M, molecular mass markers in kilodaltons. Lane 2 contains unincubated CMH-1 sample. C, samples from B were diluted 1000-fold to give a CMH-1 concentration of 2 nM and were then incubated for 30 min at 37 °C with in vitro translated S-labeled PARP in the presence or absence of Ac-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* the cleavage product. Lane B, buffer control. D, (His)(6)-CMH-1 was incubated with purified granzyme B as in B. Reactions were stopped at the indicated times and analyzed by Coomassie Blue staining.



Processing of CMH-1 in Vitro Involves Cleavage by Granzyme B and Autocatalysis

To investigate the cleavage and activation of CMH-1 by granzyme B in more detail, we incubated CMH-1 protein with granzyme B and analyzed the cleavage products by SDS-PAGE and Coomassie Blue staining. Incubation of CMH-1 with granzyme B resulted in the disappearance of the p37 and p32 peptides with the concomitant appearance of three peptides of approximately 20 (p20), 12 (p12), and 5 kDa (p5) (Fig. 3, B and D; see also Fig. 4and Fig. 5). N-terminal amino acid sequencing revealed that p20 has the same N terminus as p32 (Ala) while the p12 starts at Ser, which is also preceded by an aspartate residue (Asp). These results indicate that CMH-1 is cleaved at both Asp-Ala and Asp-Ser. We propose that the p20 and p12 polypeptides represent the two subunits of the mature protease and that the p5 peptide represents the (His)(6)-tagged CMH-1 prosequence ending at Asp.


Figure 4: Cleavage of C186S CMH-1 protein by granzyme B. Purified (His)(6)-tagged C186S (lanes 2-5 and 8-11) or wild type (lanes 6 and 7) CMH-1 were incubated for 1 h at 37 °C with granzyme B or buffer in the presence or absence of Ac-DEVD-CHO as in Fig. 3B and then analyzed by SDS-PAGE and Coomassie Blue staining (lanes 1-7) or by immunoblotting with an anti-(His)(6) antibody (lanes 8-11). Lane 1 contains C186S CMH-1 that has not been incubated.




Figure 5: Cleavage by granzyme B and activity of CMH-1 processing site mutants. A, purified wild type (lanes 7 and 8), D23E (lanes 3-6), or D198E (lanes 10-13) mutant CMH-1 proteins were incubated at 37 °C for 1 h with granzyme B or buffer in the presence or absence of Ac-DEVD-CHO as in Fig. 3B and analyzed by SDS-PAGE and Coomassie Blue staining. Lanes 2 and 9 contain unincubated D23E and D198E samples, respectively. B, samples from A were diluted 1000-fold, and their PARP cleavage activity was determined as described in Fig. 3C.



To determine if granzyme B cleaves both Asp-Ala and Asp-Ser of CMH-1 or if CMH-1 activity is involved in the processing of itself, we included the potent CMH-1 inhibitor Ac-DEVD-CHO in the reaction. In the presence of the inhibitor, the 5-kDa peptide no longer appeared (Fig. 3B and 4). Instead, a peptide of 25 kDa (p25) became visible and accumulated over time (Fig. 3, B and D). Immunoblotting indicated that this peptide contained the (His)(6) tag (Fig. 3B) suggesting that it is the unprocessed p5-p20 peptide (Fig. 3A). When we examined the cleavage products at various time points following granzyme B addition, we found that in the absence of the inhibitor, p25 initially appeared and then disappeared (Fig. 3D). The simplest interpretation of these results is that granzyme B cleaves the full-length CMH-1 p37 (and p32) between Asp and Ser to form p25 and p12 (p20 and p12), which leads to an increased autocleavage at Asp-Ala of p25 to form p20 and p5.

To further confirm that cleavage between p20 and p5 is mediated solely by autocatalytic activity of CMH-1, we expressed a mutant CMH-1 in which the putative active site cysteine in the conserved QACRG motif was changed to serine (C186S) and examined its cleavage by granzyme B (Fig. 4). First, we found that the purified C186S CMH-1 protein contained a single major polypeptide of 37 kDa, consistent with p32 in the wild type CMH-1 preparation being an autocatalytic product. Upon incubation of C186S CMH-1 with purified granzyme B, the 37-kDa peptide was cleaved into p25 and p12 with little p20 and p5. These results establish that p20 and p5 are derived from p25 and that this cleavage is mediated solely by autocatalysis. A small amount of an approximately 31-kDa peptide was also present in the C186S CMH-1 preparation and was apparently cleaved into a 19-kDa peptide upon incubation with granzyme B. The nature of this peptide was not further examined.

CMH-1 Activation Only Requires Cleavage between p20 and p12

To establish the proteolytic maturation sites in CMH-1 and more importantly to investigate the role of CMH-1 processing in its activation, we prepared mutant CMH-1 proteins with either Asp or Asp mutated to glutamate (D23E or D198E). Purified D198E protein contained two polypeptides (p37 and p32) similar to the wild type CMH-1 preparation, although there appeared to be slightly more p37 than p32 in the mutant protein. Incubation of D198E CMH-1 with granzyme B did not affect the mobility of the polypeptides indicating that Asp is the only site in CMH-1 that is recognized by granzyme B (Fig. 5A). Furthermore, granzyme B-treated D198E protein at nanomolar concentrations did not show any detectable PARP cleavage activity (Fig. 5B), indicating that the proteolytic processing between p20 and p12 at position Asp is essential for PARP cleavage activity of CMH-1.

In contrast, purified D23E protein contained mainly the 37-kDa peptide with a small amount of p25 and p12, consistent with the notion that the p32 in the wild type CMH-1 preparation is derived from p37 by autocleavage at Asp. As expected, incubation of D23E CMH-1 with granzyme B resulted in the cleavage of p37 into p25 and p12 (Fig. 5A). A small amount of p20 also appeared after incubation whether granzyme B was present or not, and its appearance was blocked by Ac-DEVD-CHO, indicating that it is a product of an autocatalytic event. This may be due to the presence of an alternative processing site near Asp, or the autocatalytic activity of CMH-1 may be capable of cleaving Glu at the P1 position albeit with lower efficiency.

Finally, we investigated if cleavage at Asp is required for CMH-1 activity. Purified wild type or D23E CMH-1 proteins were treated with granzyme B and then incubated with PARP produced by in vitro translation. As shown in Fig. 5B, at equal concentrations both the wild type and D23E CMH-1 cleaved PARP to a similar extent. In addition, an untreated D23E preparation (Fig. 5A, lane 2) showed some activity against PARP while the untreated wild type CMH-1 did not. This was consistent with the presence of a small amount of p25 and p12 in the D23E but not in the wild type CMH-1 preparation. Furthermore, unincubated D23E CMH-1 containing p25 but little p20 cleaved PARP in a similar manner as the preincubated sample, which contained p25 and p20, suggesting that p25/p12 is as active as the fully processed p20/p12 protease (compare lanes 2 and 3 in Fig. 5A). These results indicate that activation of CMH-1 requires cleavage between p20 and p12 at position Asp, whereas the cleavage between p20 and p5 is not required.

CMH-1 was recently identified based on its sequence homology to the ICE/Ced-3 family of proteases(13, 14, 15) . It has been shown that CMH-1 is processed during Fas- and tumor necrosis factor alpha-induced apoptosis (15) . Although the physiological function of CMH-1 remains unclear, its ability to cleave PARP and induce apoptosis suggests that it may play a role in cell death. In this report, we demonstrate that CMH-1 can be cleaved and activated by granzyme B in vitro, suggesting that CMH-1, a protease localized in the cytoplasm(15) , may be involved in a protease cascade that mediates CTL-induced cell killing in vivo. Our findings that CMH-1 is cleaved 25-50 times more efficiently than CPP32 by granzyme B implies that CMH-1 may be a primary target for granzyme B. Furthermore, the close homology between CMH-1 and CPP32 suggests that the mechanism of granzyme B-mediated CMH-1 activation may be applicable to CPP32. Our finding that the removal of the prosequence is not required for CMH-1 activation in vitro points to the possibility that the prosequence may be involved in other aspects of CMH-1 regulation in vivo.


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.

§
To whom correspondence should be addressed: Vertex Pharmaceuticals Inc., 40 Allston St., Cambridge, MA 02139. Tel.: 617-577-6635; Fax: 617-577-6645; su{at}vpharm.com.

(^1)
The abbreviations used are: CTL, cytotoxic T lymphocyte; ICE, interleukin-1beta-converting enzyme; PARP, poly(ADP-ribose) polymerase; PAGE, polyacrylamide gel electrophoresis; Ac-DEVD-CHO, Ac-Asp-Glu-Val-Asp-aldehyde; Ac-YVAD-CHO, Ac-Tyr-Val-Ala-Asp-aldehyde.


ACKNOWLEDGEMENTS

We thank Brett O'Hare for DNA sequencing and oligonucleotide synthesis; Drs. M. Mullican and S. Harbeson for tetrapeptide aldehydes; and Drs. J. Boger, V. Sato, and D. Livingston for helpful comments.


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