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Partial Purification and Characterization of Two Distinct Types of Caspases from Human Epidermis

1998; Elsevier BV; Volume: 111; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.1998.00295.x

1523-1747

T. Takahashi, Masashi Ogo, Toshihiko Hibino,

Autophagy in Disease and Therapy

Recent observations demonstrated that interleukin-1β converting enzyme family proteases, now referred to as caspase family, play central roles in apoptosis, or programmed cell death. In this study, we tried to isolate and characterize epidermal caspases. By DEAE-Sephacel anion-exchange chromatography, human cornified cell extract showed two caspase-like fractions (F-I and F-II) with different substrate specificities. These were further purified by Sephacryl S-200, Mono Q ion exchange and Superose 6 gel chromatography. F-I showed a molecular weight of 30 kDa and specifically hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, a fluorogenic substrate for caspase-3 (CPP32) with a Km value of 13.8 μM. F-I generated a characteristic 85 kDa fragment from poly(ADP-ribose) polymerase. Inhibitor susceptibility of F-I was very similar to that of caspase-3, further confirming the caspase-3-like properties of F-I. In contrast, the molecular weight of F-II was estimated to be 110 kDa, which was much higher than the other caspases. F-II equally hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, and acetyl-Tyr-Val-Ala-Asp-methylcoumarinamide, caspase-1 (interleukin-1β converting enzyme)-specific substrate, and was inhibited by acetyl-Tyr-Val-Ala-Asp-aldehyde and acetyl-Tyr-Val-Ala-Asp-aldehyde. Affinity labeling using biotinylated YVAD-cmk demonstrated several positive bands ranging from 25 to 35 kDa, supporting the hypothesis that F-II is a complex of multiple caspases. Reverse transcriptase-polymerase chain reaction analysis demonstrated that among known caspases tested, caspase-1, -2, -3, -4, and -7 were expressed in cultured human keratinocytes. These suggest that multiple caspases are synthesized in human keratinocytes and are involved in terminal differentiation. Recent observations demonstrated that interleukin-1β converting enzyme family proteases, now referred to as caspase family, play central roles in apoptosis, or programmed cell death. In this study, we tried to isolate and characterize epidermal caspases. By DEAE-Sephacel anion-exchange chromatography, human cornified cell extract showed two caspase-like fractions (F-I and F-II) with different substrate specificities. These were further purified by Sephacryl S-200, Mono Q ion exchange and Superose 6 gel chromatography. F-I showed a molecular weight of 30 kDa and specifically hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, a fluorogenic substrate for caspase-3 (CPP32) with a Km value of 13.8 μM. F-I generated a characteristic 85 kDa fragment from poly(ADP-ribose) polymerase. Inhibitor susceptibility of F-I was very similar to that of caspase-3, further confirming the caspase-3-like properties of F-I. In contrast, the molecular weight of F-II was estimated to be 110 kDa, which was much higher than the other caspases. F-II equally hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, and acetyl-Tyr-Val-Ala-Asp-methylcoumarinamide, caspase-1 (interleukin-1β converting enzyme)-specific substrate, and was inhibited by acetyl-Tyr-Val-Ala-Asp-aldehyde and acetyl-Tyr-Val-Ala-Asp-aldehyde. Affinity labeling using biotinylated YVAD-cmk demonstrated several positive bands ranging from 25 to 35 kDa, supporting the hypothesis that F-II is a complex of multiple caspases. Reverse transcriptase-polymerase chain reaction analysis demonstrated that among known caspases tested, caspase-1, -2, -3, -4, and -7 were expressed in cultured human keratinocytes. These suggest that multiple caspases are synthesized in human keratinocytes and are involved in terminal differentiation. acetyl-Tyr-Val-Ala-Asp-methylcouma-rinamide acetyl-Asp-Glu-Val-Asp-methylcoumarinamide acetyl-Tyr-Val-Ala-Asp-aldehyde acetyl-Tyr-Val-Ala-Asp-aldehyde biotinylated-Tyr-Val-Ala-Asp-chloromethylketone 3-[(3-cholamidopropyl) dimethylammonio] propane-sulfonic acid interleukin-1β converting enzyme poly(ADP-ribose) polymerase Apoptosis, or programmed cell death, plays important roles in various physiologic processes, such as development of organisms, tissue homeostasis, and recovery from pathologic disorders. Epidermis is one of the tissues in which apoptosis-like phenomena are often found. For example, “sunburn cells” were frequently observed in UVB-irradiated epidermis, in which nuclear condensation was a characteristic feature of these cells (Young, 1987Young A.R. et al.The sunburn cell.Photodermatol. 1987; 4: 127-134PubMed Google Scholar;Schwarz et al., 1995Schwarz A. Bhardwaj R. Aragane Y. et al.Ultraviolet-B- induced apoptosis of keratinocytes: evidence for partial involvement of tumor necrosis factor-alpha in the formation of sunburn cells.J Invest Dermatol. 1995; 104: 922-927Crossref PubMed Scopus (243) Google Scholar). Regression phase of hair follicles (catagen) was thought to be an apoptotic process (Seiberg et al., 1995Seiberg M. Marthinuss J. Stenn K.S. et al.Changes in expression of apoptosis-associated genes in skin mark early catagen.J Invest Dermatol. 1995; 104: 78-82Crossref PubMed Scopus (81) Google Scholar;Lindner et al., 1997Lindner G. Botchkarev V.A. Botchkareva N.V. Ling G. van der Veen C. Paus R. et al.Analysis of apoptosis during hair follicle regression (catagen).Am J Pathol. 1997; 151: 1601-1607PubMed Google Scholar). Moreover, terminal differentiation of keratinocytes is suggested to be an elaborative pathway of apoptosis because it includes nuclear condensation and destruction of nuclei (McCall and Cohen, 1991McCall C.A. Cohen J.J. et al.Programmed cell death in terminally differentiating keratinocytes: role of endogenious endonuclease.J Invest Dermatol. 1991; 97: 111-114Abstract Full Text PDF PubMed Google Scholar;Haake and Polakowska, 1993Haake A.R. Polakowska R.R. et al.Cell death by apoptosis in epidermal biology.J Invest Dermatol. 1993; 101: 107-112Abstract Full Text PDF PubMed Google Scholar;Polakowska et al., 1994Polakowska R.R. Piacentini M. Bartlett R. Goldsmith L.A. Haake A.R. et al.Apoptosis in human skin development: morphogenesis, periderm, and stem cells.Dev Dyn. 1994; 199: 176-188Crossref PubMed Scopus (242) Google Scholar). The molecular mechanism of epidermal apoptosis has not been well understood, however. Detailed studies on genetic pathways of programmed cell death in nematode C. elegance demonstrated that ced-3 was essential for the execution of the cell death program (Yuan and Holvitz, 1990Yuan J. Holvitz H.R. et al.Genetic mosaic analyses of ced-3 and ced-4, two genes that control programmed cell death in the nematode C. elegance.Dev Biol. 1990; 138: 33-41Crossref PubMed Scopus (432) Google Scholar). Cloning of ced-3 gene showed significant sequence homology to the mammalian cysteine protease, interleukin 1-β converting enzyme (ICE) (Yuan et al., 1993Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. The C. et al.elegance cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme.Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2177) Google Scholar). In addition, over-expression of ICE was found to induce apoptotic cell death in rat fibroblasts (Miura et al., 1993Miura M. Zhu H. Rotello R. Hartwieg E.A. Yuan J. et al.Induction of apoptosis in fibroblasts by IL-1 beta converting enzyme, a mammalian homologue of the C. elegance cell death gene ced-3.Cell. 1993; 75: 653-660Abstract Full Text PDF PubMed Scopus (1304) Google Scholar). The fact that inhibition of ICE activity by a potent ICE inhibitor, cytokine response modifier A (CrmA) from cowpox virus (Ray et al., 1992Ray C.A. Black R.A. Kronheim S.R. Greenstreet T.A. Sleath P.R. Salvesen G.S. Pickup D.J. et al.Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1β converting enzyme.Cell. 1992; 69: 597-604Abstract Full Text PDF PubMed Scopus (868) Google Scholar), prevented apoptosis in various cells (Miura et al., 1993Miura M. Zhu H. Rotello R. Hartwieg E.A. Yuan J. et al.Induction of apoptosis in fibroblasts by IL-1 beta converting enzyme, a mammalian homologue of the C. elegance cell death gene ced-3.Cell. 1993; 75: 653-660Abstract Full Text PDF PubMed Scopus (1304) Google Scholar;Gagliardini et al., 1994Gagliardini V. Fernandez P-A Lee R.K.K. Drexler H.C.A. Rotello R.J. Fishman M.C. Yuan J. et al.Prevention of vertebrate neuronal death by the crmA gene.Science. 1994; 263: 826-828Crossref PubMed Scopus (596) Google Scholar;Tewari and Dixit, 1995aTewari M. Dixit V.M. et al.Fas- and tumor necrosis factor-induced apoptosis is inhibited by the poxvirus crmA gene product.J Biol Chem. 1995 a; 270: 3255-3260Crossref PubMed Scopus (591) Google Scholar) further supported critical roles of ICE in higher organisms. A number of attempts to hunt for enzymes similar to ICE have been reported elsewhere, and 10 human ICE-related genes have been cloned until recently. Such ICE-like proteases were classified as ICE/ced-3 family, which was recently named caspase family (Alnemri et al., 1996Alnemri E.S. Livingstone D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Human ICE/CED-3 protease nomenclature.Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2077) Google Scholar). On the other hand, very limited reports were available for the purification of active caspases from cells and tissues. Active caspase-1 (ICE) has been purified from human monocyte THP.1 cells (Thornberry et al., 1992Thornberry N.A. Bull H.G. Calaycay J.R. et al.A novel heterodimeric cysteine protease is required for interleukin-1β processing in monocytes.Nature. 1992; 356: 768-774Crossref PubMed Scopus (2088) Google Scholar;Miller et al., 1993Miller D.K. Ayala J.M. Egger L.A. et al.Purification characterization of active human interleukin-1 beta-converting enzyme from THP. 1 monocyte cells.J Biol Chem. 1993; 268: 18062-18069Abstract Full Text PDF PubMed Google Scholar). Caspase-3 (CPP32/YAMA/apopain), which was identified as one of the ICE homologs (Fernandes-Alnemri et al., 1994Fernandes-Alnemri T. Litwack G. Alnemri E.S. CPP 32, a novel human apoptotic protein with homology to caenorhabditis elegance cell death protein Ced-3 and mammalian interleukin-1 beta converting enzyme.J Biol Chem. 1994; 269: 30761-30764Abstract Full Text PDF PubMed Google Scholar;Tewari et al., 1995bTewari M. Quan L.T. O’rourke K. et al.Yama/CPP 32 beta, a mammalian homologue of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly (ADP-ribose) polymerase.Cell. 1995 b; 81: 801-809Abstract Full Text PDF PubMed Scopus (2209) Google Scholar), has been purified from THP.1 cells as a protein responsible for cleavage of poly(ADP-ribose) polymerase (PARP) during apoptosis (Nicholson et al., 1995Nicholson D.W. Ali A. Thornberry N.A. et al.Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3682) Google Scholar). And also, Pai et al. have partially purified two distinct types of proteases that cleave sterol-regulatory element binding protein (SREBP) from hamster liver. Peptide sequence analysis revealed that they were identical to caspase-3 and caspase-7 (Mch3/ICE-LAP3/CMH-1), respectively (Pai et al., 1996Pai J-T Brown M.S. Goldstein J.L. et al.Purification and cDNA cloning of a second apoptosis-related cysteine protease that cleaves and activates sterol regulatory element binding proteins.Proc Natl Acad Sci. 1996; 93: 5437-5442Crossref PubMed Scopus (63) Google Scholar). In this study, we tried to investigate participation of caspase-like proteases in keratinocyte differentiation. The involvement of caspases in proliferation and differentiation of keratinocytes is still being debated. Here we describe the partial purification and biochemical characterization of epidermal caspases. We successfully obtained two distinct types of caspase-like proteases from normal human cornified cell extract. These two fractions, which are referred to as F-I and F-II, were quite different from each other in terms of molecular weight, substrate specificity, and inhibitor susceptibility, yet both hydrolyzed PARP with the cleavage product of 85 kDa fragment. Our results suggest that multiple types of caspases participate in the epidermal apoptosis and would be related to the terminal differentiation of keratinocytes. Synthetic fluorogenic tetrapeptide substrates [acetyl-Tyr-Val-Ala-Asp-methylcoumarinamide (Ac-YVAD-MCA), acetyl-Asp-Glu-Val-Asp-methylcoumarinamide (Ac-DEVD-MCA)], tetrapeptide inhibitors [acetyl-Tyl-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO), acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO)], and protease inhibitors (E-64 and pepstatin A) were purchased from Peptide Institute (Osaka, Japan). Biotinyl-Tyl-Val-Ala-Asp-chrolomethylketone (biotin-YVAD-cmk) was a product of Calbiochem (La Jolla, CA). Protease inhibitors phenylmethylsulfonyl fluoride and N-ethylmaleimide were obtained from Wako Pure Chemicals (Tokyo, Japan). Tosyl-lysine chloromethylketone, aprotinin, leupeptin, and antipain were from Boehringer (Mannheim, Germany), and iodoacetic acid, soybean trypsin inhibitor were from Sigma (St. Louis, MO). DEAE-Sephacel, Sephacryl S-200, FPLC Mono Q, and Precision Column Superose 6 for SMART system were purchased from Pharmacia (Uppsala, Sweden). All other chemicals were of reagent grade. Caspase activity was measured using fluorogenic substrates Ac-YVAD-MCA or Ac-DEVD-MCA (Miller et al., 1993Miller D.K. Ayala J.M. Egger L.A. et al.Purification characterization of active human interleukin-1 beta-converting enzyme from THP. 1 monocyte cells.J Biol Chem. 1993; 268: 18062-18069Abstract Full Text PDF PubMed Google Scholar). Briefly, 50 μl of samples were incubated at 37°C for 30 min with 50 μM of substrate in 150 μl of 25 mM HEPES containing 10% sucrose, 0.1% 3-[(3-cholamidopropyl) dimethylammonio] propane-sulfonic acid (CHAPS) and 1 mM dithiothreitol (HSCD buffer). The reaction was stopped by adding 2 ml of 0.1 M chloroacetic acid. Liberation of aminomethylcoumarin was measured by the fluorescence intensity at 460 nm excited by 380 nm recorded on a FP-777 fluorophotometer (JASCO, Tokyo, Japan). Inhibitor assays were performed similarly by adding 50 μl of inhibitor samples. Inhibitor susceptibility was estimated by the residual caspase activity after incubation at 37°C for 30 min using phosphate-buffered saline as a control. Protein concentration was determined by bicinchonic acid (BCA) method with BCA Protein Assay Reagent (Pierce, Rockford, IO) using bovine serum albumin as a standard. All purification steps were carried out at 4°C. Human cornified cells (30 g) scraped from heels of healthy volunteers (age 27–42) were homogenized in 0.1 M Tris-HCl, pH 8.0 containing 0.14 M NaCl, 0.1% CHAPS, and 1 mM dithiothreitol with a glass homogenizer (Wheaton, Millville, NJ). The resulting suspension was extracted for 2 h. After centrifugation at 25,000 ×g for 15 min, the supernatant was filtered with a bottle-top filter unit (0.22 μm) equipped with a glass fiber prefilter. The filtrate was concentrated about 10-fold by ultrafiltration using YM-10 membrane (Amicon, Beverly, MA) and dialyzed overnight against 20 mM Tris-HCl, pH 7.8 containing 10% sucrose, 0.1% CHAPS, and 1 mM dithiothreitol (buffer A). The crude extract was applied to a DEAE-Sephacel ion-exchange column (2.5 cm × 20 cm), equilibrated with buffer A, and was eluted with a two-step linear gradient of NaCl from 0 to 0.3 M and from 0.3 M to 1 M. Enzymatically active fractions were pooled, concentrated, and applied onto a Sephacryl S-200 gel filtration column (1.6 × 90 cm) equilibrated with phosphate-buffered saline containing 10% sucrose, 0.1% CHAPS, and 1 mM dithiothreitol (buffer B). The active fractions were concentrated to 2.5 ml and passed through a PD-10 column equilibrated with buffer A. The enzyme fractions were applied onto a Mono Q fast protein liquid chromatography column and eluted with increasing NaCl concentration up to 0.5 M. The enzyme fractions were collected and concentrated for further analysis. Finally, caspase-like enzymes were further purified by Superose 6 gel filtration using SMART system to determine their molecular weights using LMW Gel Filtration Calibration Kit (Pharmacia) consisting of blue dextran (>2 × 106), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa). Enzyme samples in HSCD buffer were incubated with various concentrations of fluorogenic substrates. Initial rate v was calculated from the increase of the liberated aminomethylcoumarin, and Michaelis constant Km was determined by the plots of 1/[S] and 1/v (Lineweaver–Burk plots). Inhibition constant Ki was determined from the plots of [I] and 1/v with different substrate concentrations (Dixon plots). PARP was purified from bovine thymus according to the method ofYoshihara et al., 1978Yoshihara K. Hashida T. Tanaka Y. Ohgushi H. Yoshihara H. Kamiya T. et al.Bovine thymus poly (adenosine diphosphate ribose) polymerase.J Biol Chem. 1978; 253: 6459-6466Abstract Full Text PDF PubMed Google Scholar. Purified bovine PARP was incubated with enzyme fractions in HSCD buffer for 30 min at 37°C. Cleavage products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent western blotting. After SDS-PAGE, proteins were transferred to a polyvinylidenefluoride membrane (Millipore, Bedford, MA) at 2 mA per cm2 for 2 h. The membranes were immersed in 3% skim milk (Snow Brand, Sapporo, Japan), and incubated with anti-PARP antibody (Enzyme System Products, Dublin, CA), followed by horseradish peroxidase conjugated anti-rabbit IgG. Enhanced Chemiluminescence (ECL) Western Blotting Detection System (Amersham, Bucks, U.K.) was used for detection. Molecular weight of the cleavage product was determined by Prestained SDS-PAGE Standards (BioRad, Hercules, CA). Biotin-labeled YVAD-cmk was used as an affinity ligand to detect caspase-like enzymes. The purified protease samples were incubated with the biotin-YVAD-cmk for 30 min at room temperature, and applied onto 12.5% SDS or native-PAGE gels under nonreducing condition. After transfer to a polyvinylidenefluoride membrane, the enzyme-inhibitor complexes were stained with horseradish peroxidase-conjugated streptavidin (BioRad) and visualized using ECL detection system. Molecular weight was estimated using Biotinylated SDS-PAGE Standards (BioRad). Human keratinocytes (Kurabo, Osaka, Japan) were cultured in a serum-free K-GM medium. Poly (A+) RNA were isolated from the cultured keratinocytes at 50%, 80%, 100% confluency as well as 2 d after confluency (namely 120%) using FastTrack mRNA Isolation Kit (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. For the synthesis of oligo-dT-primed cDNA, 250 ng of poly (A+) RNA was reverse transcripted using 200 Units of SuperScript II (Gibco/BRL, Gaithersburg, MD) primed by 500 ng of oligo-(dT)15 primers (Promega, Madison, WI), in a total volume of 20 μl. After 1 h incubation at 37°C, the reaction was stopped by heating at 95°C for 5 min, and 1 μl of reverse transcriptase products were used as templates for each PCR reaction. The PCR primers of seven caspase family members (caspase-1–7) were designed based on the published mRNA sequences: caspase-1, 5′-CTGCCCAAGTTTGAAGGA-3′ and 5′-TTTAATGTCCTGGGAAG-3′ (expected length 310 bp); caspase-2 (Ich-1), 5′-ATGGCCGCTGACAGGGG-3′ and 5′-GAACAGAAACCGTGCAT-3′ (1080 bp); caspase-3, 5′-ATACTCCTTCCATCAAATAG-3′ and 5′-AACATCACAAAACCATAATC-3′ (410 bp); caspase-4 (TX/ICH-2/ICErel-II), 5′-GCTGTTTACAAGACCCACGTGG-3′, and 5′-GTGGCTTCCATTTTCAA-3′ (285 bp); caspase-5 (ICErel-III/TY), 5′-TCTGTTTGCAAGATCCACGAGA-3′ and 5′-TGTGGTTTCATTTTCAA-3′ (279 bp); caspase-6 (Mch2), 5′-ATGAGCTCGGCCTCGGG-3′ and 5′-TAGCCTCTATTAATTAA-3′ (915 bp); caspase-7, 5′-ATGGCAGATGATAGGGC-3′ and 5′-GACCCATTGCTTCTCAGC-3′ (917 bp). PCR amplification was performed for 35 cycles (40 s at 94°C, 50 s at 50–55°C, 40 s at 72°C). PCR products were analyzed on 2% agarose gel electrophoresis. Figure 1 shows an elution profile of DEAE-Sephacel chromatography. Two active fractions of caspase-like enzymes were eluted at ≈0.25 and 0.30 M NaCl concentrations. These are designated as F-I and F-II, respectively. F-I showed a hydrolytic activity on Ac-DEVD-MCA, a substrate designed for caspase-3, but had little effect on Ac-YVAD-MCA, a substrate for caspase-1. F-I was further purified by Sephacryl S-200 gel column and FPLC Mono Q, respectively. By Sephacryl S-200, F-I showed a DEVD-specific activity at a molecular weight of 30 kDa, and by Mono Q chromatography at pH 7.8, F-I was eluted at ≈0.25 M NaCl (data not shown). On the other hand, F-II hydrolyzed both Ac-DEVD-MCA and Ac-YVAD-MCA to a similar extent. F-II from DEAE-Sephacel chromatography was further purified by the same steps. When we applied F-II to Sephacryl S-200 gel chromatography, caspase-like activity eluted at much higher molecular weight than 30 kDa (data not shown). To determine the molecular weight of these enzyme fractions, we used a SMART system equipped with a Superose 6 gel filtration column. Figure 2 shows the elution profiles of F-I and F-II by Superose 6 chromatography, respectively. F-I showed a sharp and single peak at a molecular weight of 30 kDa (Figure 2a). Molecular weight of F-II was estimated to be ≈110 kDa (Figure 2b), which was much higher than those of caspase family members. F-II demonstrated hydrolytic activities on both Ac-YVAD-MCA and Ac-DEVD-MCA substrates. We used partially purified F-I and F-II from the Mono Q step as samples for biochemical and enzymologic characterization. Kinetic constants were determined for F-I and F-II using tetrapeptide substrates with a continuous fluorometric assay as shown in Table I. Cleavage activity of Ac-DEVD-MCA by F-I showed Michaelis–Menten kinetics with a Km value of 13.8 μM. F-I did not show any detectable activity on Ac-YVAD-MCA in this assay condition. The Ki value against Ac-DEVD-CHO is 0.0389 nM, which means that Ac-DEVD-CHO is a very specific inhibitor for F-I. F-II demonstrated different kinetic properties from the known caspases. Although it cleaved both tetrapeptide substrates, Km values for Ac-DEVD-MCA and Ac-YVAD-MCA were 402 μM and 1029 μM, respectively, which were considerably higher than the values of F-I. The Ki value against Ac-YVAD-MCA was 8.19 μM, which shows that Ac-YVAD-CHO is a relatively weak inhibitor for F-II. Table II summarizes the result of inhibitor susceptibility for F-I and F-II. F-I was inhibited by thiol-alkylating reagents, N-ethylmaleimide and iodoacetic acid. On the other hand, E-64, which was also a universal cysteine protease inhibitor, did not show any effect on F-I. Other serine, aspartic, and metallo-protease inhibitors had no effect on F-I (Table II). F-II showed unusual inhibitor susceptibility compared with F-I, though it retained basic nature as a caspase. F-II was almost insusceptible at 1 mM concentration of N-ethylmaleimide and iodoacetic acid, whereas more than 90% of F-I activity was inhibited. Even at 0.1 M N-ethylmaleimide and iodoacetic acid, ≈30% of F-II activity was observed. When tetrapeptide inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO were used, F-I and F-II showed different inhibition profiles as shown in Figure 3. The activity of F-I was not inhibited by Ac-YVAD-CHO up to 10 μM, but was inhibited by Ac-DEVD-CHO in a concentration-dependent manner. F-II was inhibited by both inhibitors to the similar extent; however, inhibition was rather weak compared with F-I. Because caspase-3 was identified as an enzyme responsible for PARP inactivation during apoptosis, we examined PARP cleavage activity using purified bovine PARP as a substrate. Western blot analysis (Figure 4) showed cleavage product at 85 kDa after incubation with F-I, supporting the hypothesis that F-I is a caspase-3-like protease. When F-II was incubated with the purified PARP, it was also able to generate 85 kDa fragment, showing that F-II shared a caspase-3-like property. The pH dependence of F-I and F-II was measured at various pH values using Ac-DEVD-MCA as a substrate. As shown in Figure 5, both fractions were most active at pH 7.5. F-II, however, showed broader pH optimum. F-II showed more than 50% activity even at pH 5.5 compared with that of F-I at pH 7.5, whereas F-I showed sharp pH dependence. These data suggest that F-II is the most unusual type of caspase ever identified. For further evaluation of F-II, affinity-labeled complex was analyzed by horseradish peroxidase-conjugated streptavidin after incubation with large excess of biotin-YVAD-cmk for 30 min. In native-PAGE, F-II-YVAD complex was detected in a single band at high molecular weight region (Figure 6, lane 1). On the other hand, SDS-PAGE did not demonstrate any band at high molecular weight, but several bands were observed in the 25–35 kDa range (Figure 6, lane 2). These results suggest that F-II is a complex consisting of different species of caspase family members, and the complex is dissociable in the presence of SDS. We designed specific primer pairs for seven caspase family members, caspase-1 to caspase-7, based on the published cDNA sequences. As shown in Figure 7, mRNA of caspase-1 (310 bp), -2 (1080 bp), -3 (410 bp), -4 (285 bp), and -7 (917 bp) were detected in cultured human keratinocytes at different stages. Interestingly, expression levels of each caspase showed significant difference. After the cells reached confluency, expression of caspase-7 was no longer detectable. Quantities of amplified cDNA of caspase-1, caspase-2, and caspase-3 were gradually decreased, whereas caspase-4 did not show significant changes and maintained its expression level after the confluent condition. Although apoptosis-like phenomena are often found in human epidermis, participation of caspases has not been reported to date. Thus, it is important to purify and identify the epidermal caspases. This study isolates caspase-like proteases from normal epidermis. Two distinct types of caspase-like proteases (F-I and F-II) were obtained from human cornified cell extract, which were iodoacetic acid and N-ethylmaleimide-sensitive but E-64-insensitive cysteine proteases. This kind of selective inhibitor susceptibility to general cysteine protease inhibitors is a characteristic feature for caspases (Thornberry et al., 1992Thornberry N.A. Bull H.G. Calaycay J.R. et al.A novel heterodimeric cysteine protease is required for interleukin-1β processing in monocytes.Nature. 1992; 356: 768-774Crossref PubMed Scopus (2088) Google Scholar;Nicholson et al., 1995Nicholson D.W. Ali A. Thornberry N.A. et al.Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3682) Google Scholar); however, F-I and F-II showed considerably different properties in terms of activity, molecular weight, substrate specificity, and inhibitor susceptibility. Among human caspases, caspase-3, caspase-6, and caspase-7 possess similar specificity to tetrapeptide DEVD substrate and designated as caspase-3 subfamily (Duan et al., 1996Duan H. Chinnaiyan A.M. Hudson P.L. Wing J.P. He W-W Dixit V.M. ICE-LAP3, a novel mammalian homologue of the Caenorhabditis elegans cell death protein Ced-3 is activated during Fas- and tumor necrosis factor-induced apoptosis.J Biol Chem. 1996; 271: 1621-1625Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). The low molecular weight fraction, F-I, showed almost identical properties to those of the caspase-3 subfamily. F-I specifically hydrolyzed Ac-DEVD-MCA and was inhibited by Ac-DEVD-CHO. F-I did not show any effect on Ac-YVAD-MCA, and Ac-YVAD-CHO had no effect on F-I activity. Molecular weight estimated by gel filtration is identical to the heterodimeric complex of p20 and p10 (Thornberry et al., 1992Thornberry N.A. Bull H.G. Calaycay J.R. et al.A novel heterodimeric cysteine protease is required for interleukin-1β processing in monocytes.Nature. 1992; 356: 768-774Crossref PubMed Scopus (2088) Google Scholar). The Km value of F-I (13.8 μM) is comparable with those of 9.7 μM for caspase-3 from THP.1 cells (Nicholson et al., 1995Nicholson D.W. Ali A. Thornberry N.A. et al.Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3682) Google Scholar), 13 μM of recombinant caspase-3, and 51 μM of recombinant caspase-7 (Fernandes-Alnemri et al., 1995Fernandes-Alnemri T. Takahashi A. Armstrong R. et al.Mch3, a novel human apoptotic cysteine protease highly related to CPP 32.Cancer Res. 1995; 55: 6045-6052PubMed Google Scholar). The Ki value (0.0389 nM) is also similar to that of caspase-3, which was lower than 1 nM (Nicholson et al., 1995Nicholson D.W. Ali A. Thornberry N.A. et al.Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3682) Google Scholar). These kinetic parameters strongly suggest that F-I is a caspase-3-like protease. Proteolytic cleavage of PARP is a distinctive phenomenon during apoptosis and it is induced by a protease resembling ICE, but not by ICE itself (Lazebnik et al., 1994Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. et al.Cleavage of poly (ADP-ribose) polymerase by a proteinase with properties like ICE.Nature. 1994; 371: 346-347Crossref PubMed Scopus (2284) Google Scholar). Recent studies have shown that protease resembling ICE is a member of the caspase-3 subfamily. Our results also revealed that F-I possessed a limited proteolytic activity on PARP and generated a specific 85 kDa cleavage product. Taken together, F-I is a caspase-3-like cysteine protease. F-II, on the other hand, has distinct properties compared with other caspases. F-II was not obtained when cornified cells were extracted in the absence of CHAPS (data not shown), thus, the addition of CHAPS is essential for the extraction of F-II. This result suggests that F-II is associated with cell membrane or contains membrane-bound proteins. Although F-II is also an E-64-insensitive cysteine protease, its properties are quite different from other caspases. It hydrolyzed both YVAD and DEVD tetrapeptide substrates with considerably lower affinity, and showed unusually higher molecular weight. The Km values for Ac-DEVD-MCA and Ac-YVAD-MCA were considerably higher than 9.7 μM for caspase-3 (Nicholson et al., 1995Nicholson D.W. Ali A. Thornberry N.A. et al.Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3682) Google Scholar) and 14.3 μM for caspase-1 (Thornberry et al., 1992Thornberry N.A. Bull H.G. Calaycay J.R. et al.A novel heterodimeric cysteine protease is required for interleukin-1β processing in monocytes.Nature. 1992; 356: 768-774Crossref PubMed Scopus (2088) Google Scholar). Taken together, these results suggest that F-II is different in protease function from known caspases. These unique properties of F-II might be ascribed to a formation of hetero-oligomer consisting of different species of caspase family members. Caspase-like proteases consist of p20 and p10 subunits to form active heterodimer (Thornberry et al., 1992Thornberry N.A. Bull H.G. Calaycay J.R. et al.A novel heterodimeric cysteine protease is required for interleukin-1β processing in monocytes.Nature. 1992; 356: 768-774Crossref PubMed Scopus (2088) Google Scholar). X-ray crystallography revealed that recombinant caspase-1 protein exists in the form of tetramer consisting of two heterodimers (Walker et al., 1994Walker N.P.C. Talanian R.V. Brady K.D. et al.Crystal structure of the cysteine protease interleukin-1β-converting enzyme: a (p20/p10) 2 homodimer.Cell. 1994; 78: 343-352Abstract Full Text PDF PubMed Scopus (512) Google Scholar;Wilson et al., 1994Wilson K.P. Black J-Af Thomson J.A. et al.Structure and mechanism of interleukin-1β converting enzyme.Nature. 1994; 370: 270-275Crossref PubMed Scopus (731) Google Scholar). Because RT-PCR analysis revealed that five different types of caspases are expressed in cultured human keratinocytes, and affinity labeling proved association of several 30 kDa species to form oligomeric F-II, it is reasonable to postulate that multiple species of caspase family members associate each other and form hetero-oligomers. The decrease in affinity to substrates and to inhibitors can be explained by the partial denaturation during the extraction process or the involvement of inactive precursor form (45 kDa) to form hetero-dimerization with different species. Another possibility is a complex formation with unidentified binding protein to regulate caspase activity. A couple of evidences showed the hetero-oligomerization of caspases could occur, as seen in caspase-3 and caspase-7 (Fernandes-Alnemri et al., 1995Fernandes-Alnemri T. Takahashi A. Armstrong R. et al.Mch3, a novel human apoptotic cysteine protease highly related to CPP 32.Cancer Res. 1995; 55: 6045-6052PubMed Google Scholar), caspase-1 and caspase-4 (Gu et al., 1995Gu Y. Wu J. Faucheu C. Lalanne J-L Diu A. Livingston D.J. Su MS-S et al.Interleukin-1beta converting enzyme requires oligomerization for activity of processed forms in vivo.Embo J. 1995; 14: 1923-1931Crossref PubMed Scopus (130) Google Scholar). Recombinant caspase-1 subunits are in reversible equilibrium to form oligomers in vitro, showing different activity and stability (Talanian et al., 1996Talanian R.V. Dang L.C. Hackett M.C. Mankovich J.A. Welch J.P. Wong W.W. Brady K.D. et al.Stability and oligomeric equilibria of refolded interleukin-1beta converting enzyme.J Biol Chem. 1996; 272: 21853-21858Google Scholar). It also reported that C. elegance cell death factor Ced-3 can interact with Ced-4 and Ced-9 (Chinnaiyan et al., 1997Chinnaiyan A.M. O’rourke K. Lane B.R. Dixit V.M. Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death.Science. 1997; 275: 1122-1126Crossref PubMed Scopus (543) Google Scholar;Spector et al., 1997Spector M.S. Desnoyers S. Hoeppner D.J. Hengartner M.O. et al.Interaction between the C. elegance cell-death regulators CED-9 and CED-4.Nature. 1997; 385: 653-656Crossref PubMed Scopus (249) Google Scholar). Recently, human Ced-4 homolog (Apaf-1) has been isolated from cytosolic extract of HeLa cells (Zou et al., 1997Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. et al.Apaf-1 a human protein homologous to C. elegance CED-4, participates in cytochrome c-dependent activation of caspase-3.Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2649) Google Scholar). Apaf-1 forms a complex with cytochrome c and caspase-9 (Li et al., 1997Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. et al.Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (5976) Google Scholar). These observations suggest that it is quite possible to associate different caspase family members to form oligomeric protease species like F-II in this study. RT-PCR analysis demonstrated that at least five caspase mRNA were expressed in cultured human keratinocytes. Expression of caspase-3 and caspase-7 supports the presence of caspase-3-like fraction, F-I, in the cornified cell extract. Expression of multiple caspase family members supports the oligomeric feature of F-II. In addition, our results clearly showed that the production of caspases starts at very early stages when cells are still in log phase proliferation. Although we have no evidence that oligomerization is required for the induction of keratinocyte apoptosis, oligomerization of multiple caspases as seen in F-II seems to be a unique character. The presence of two distinct fractions may represent an activation process of a caspase cascade in epidermis. It has been shown that caspase-3-like activity is gradually increased depending on the caspase-1-like protease activity in Fas-mediated apoptosis (Enari et al., 1996Enari M. Talanian R.V. Wong W.W. Nagata S. Sequential activation of ICE-like and CPP 32-like proteases during Fas-mediated apoptosis.Nature. 1996; 380: 723-726Crossref PubMed Scopus (955) Google Scholar). This means that caspase-3 is activated downstream of caspase-1 in a sequential caspase cascade. Our results suggest that a similar mechanism may occur in terminal differentiation of keratinocytes. Because YVAD-specific fraction was not extracted from cornified cells, activation of caspase-3 (or caspase-3-like protease) may occur downstream of caspase-1 (or caspase-1-like protease) in terminally differentiated keratinocytes. Concerning the results of biochemical characterization, it might be possible to postulate that F-I is an activated caspase-3-like protease released from F-II, and F-II can be considered as a partially active intermediate complex in the course of the activation process as observed in the case of caspase-3 activation by Apaf complex (Li et al., 1997Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. et al.Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (5976) Google Scholar). It is now evident that the terminal differentiation of keratinocytes involves activation of caspases, in other words, apoptotic processes. Epidermal apoptosis, however, seems to be much more complicated and likely to involve more factors to form the functional barrier against the environment. Caspases extracted in this study may play important roles in the regulation of epidermal apoptosis and thus for controlling terminal differentiation.