COMMUNICATION:
Spodoptera frugiperda Caspase-1, a Novel Insect Death Protease That Cleaves the Nuclear Immunophilin FKBP46, Is the Target of the Baculovirus Antiapoptotic Protein p35*

(Received for publication, October 1, 1996, and in revised form, November 22, 1996)

Manzoor Ahmad Dagger , Srinivasa M. Srinivasula Dagger , Lijuan Wang , Gerald Litwack , Teresa Fernandes-Alnemri and Emad S. Alnemri §

From the Center for Apoptosis Research, the Department of Biochemistry and Molecular Pharmacology, and the Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Employing the degenerate primer-dependent polymerase chain reaction approach used recently to clone human Mch2, we have identified and cloned the insect Spodoptera frugiperda target of the baculovirus antiapoptotic protein p35. This protein named Sf caspase-1 belongs to the family of caspases and is highly related to human Mch3 and CPP32 in sequence and specific activity. The proenzyme of Sf caspase-1 is 299 amino acids in length and can undergo autocatalytic processing in Escherichia coli to an active enzyme heterocomplex. Autoprocessing occurs at Asp-28, Asp-184, and Asp-195 to generate the large p19/p18 and small p12 subunits. Sf caspase-1 is able to induce apoptosis in Sf9 cells and is capable of cleaving p35 to similar sized fragments as observed with extracts from p35 null mutant baculovirus-infected Sf9 cells. Sf caspase-1 activity is potently inhibited by p35, suggesting that it is an important target of this antiapoptotic protein. Finally, the Sf9 nuclear immunophilin FKBP46 was identified as a death-associated substrate for Sf caspase-1.


INTRODUCTION

Aspartate-specific cysteine proteases (caspases) (1) play a central and evolutionarily conserved role in transducing the apoptotic signal and final execution of apoptosis (2-6). In the human there are 10 different caspases, divided into three subfamilies based on their homology to the mammalian proinflammatory prototype interleukin 1beta converting enzyme (ICE)1 and the nematode proapoptotic prototype CED-3 (7, 8). In mammalian cells it is now believed that caspases with long N-terminal prodomains such as ICH-1, Mch4, and Mch5 (caspase-2, -10, and -8, respectively) might be the most upstream transducers of diverse apoptotic signals, whereas those with short prodomains such as CPP32, Mch2, and Mch3 (caspase-3, -6, and -7, respectively) are the downstream executioners of apoptosis (5, 7-9).

Studies with the baculovirus Autographa californica and its insect host Spodoptera frugiperda identified baculovirus-encoded proteins, p35 and IAPs (<UNL>i</UNL>nhibitor of <UNL>a</UNL>poptosis <UNL>p</UNL>rotein), that suppress baculovirus-induced apoptosis in S. frugiperda cells (10-12). These proteins are expressed by the baculovirus to counter the host's antiviral defense (i.e. apoptosis) to ensure virus latency and multiplication. Mammalian antiapoptotic proteins homologous to baculovirus IAPs have recently been identified (13-15). In contrast, no mammalian counterpart of p35 has yet been identified. Nevertheless, p35 is an effective suppressor of apoptosis in mammalian cells (16-18). Its antiapoptotic activity is attributed to its ability to interact with and potently inhibit members of the caspase family (19, 20). This suggests that the apoptotic program in S. frugiperda is similar to the mammalian program and is mediated by active caspase(s). This is further supported by the recent observation that baculovirus infection of S. frugiperda cells activates a caspase that can cleave p35 (21).

To identify and clone the S. frugiperda caspase that is responsible for execution of apoptosis in this organism, we employed a degenerate PCR approach designed to identify caspases in different species. Here we report the complete amino acid sequence of an S. frugiperda caspase named Sf caspase-1, as deduced from its cDNA. We demonstrate that this protease is capable of cleaving p35 and is potently inhibited by p35. This protease is also able to induce apoptosis in Sf9 cells and cleave the Sf9 nuclear immunophilin FKBP46 (22). Sf caspase-1 has a short prodomain and is related to human CPP32 and Mch3, implying that it is a downstream executioner of apoptosis.


MATERIALS AND METHODS

Cloning of Sf Caspase-1 Proenzyme

An aliquot (10 µl) of Sf9 lambda  Uni-ZAPTM XR cDNA library (22) containing ~108 plaque-forming units was denatured at 99 °C for 5 min and then subjected to two PCR amplification steps in the presence of degenerate primers encoding the pentapeptide GSWFI/GSWYI and QACRG as described (6, 23). The secondary PCR products were then used as probes to obtain full-length Sf caspase-1 clones.

Expression of Sf Procaspase-1 and p35

The open reading frame of Sf procaspase-1 was subcloned into the bacterial expression vector pET21b in-frame with an N-terminal T7-tag and a C-terminal His-tag and transformed into BL21(DE3) bacteria. The mature protease was purified on a Ni2+-affinity resin and microsequenced by automated Edman degradation (Applied Biosystems 477A equipped with a data analyzer). ProCPP32 and proMch3 were expressed and purified in a similar fashion. p35 was expressed in the same system without T7-tag. Baculovirus encoding T7-Sf procaspase-1-His6 under the late polyhedrin promoter was generated as described previously (22). Because baculovirus replication occurs early during infection, late Sf caspase-1 synthesis has no effect on baculovirus propagation.

In Vitro Transcription/Translation and Cleavage Assays

p35 and Sf caspase-1 cDNAs were in vitro transcribed and translated in the presence of [35S]methionine as described recently (7-9). Two µl of translation mixture was incubated with pure enzymes or cell extracts in a 10-µl volume for various times at 37 °C. The products were analyzed by Tricine-SDS-PAGE.

Preparation of Sf9 Apoptotic Extracts

p35 null mutant baculovirus (vp35Delta ) was propagated in TN-368 insect cells which are resistant to baculovirus-induced apoptosis. Sf9 cells were infected with wild type or vp35Delta and harvested 24 h after infection. The cells were suspended in ICE buffer (7) and lysed by a 2-3 cycle of freeze-thaw followed by homogenization. The cell lysates were centrifuged at 16,000 × g for 15 min, and the supernatants were collected and then used for the enzymatic assays.

Western Blot Analysis of Sf FKBP46 and Sf Caspase-1

Sf FKBP46 and the T7-tagged Sf caspase-1 were analyzed by Western blotting using a rabbit polyclonal antibody raised against Sf9 FKBP46 (22) or T7-antibody (Novagen), respectively.


RESULTS AND DISCUSSION

Sf Procaspase-1 Belongs to the CED-3 Subfamily of Caspases

Using a PCR approach developed recently in our laboratory to identify and clone novel members of the caspase family from different species (23), a 2.4-kilobase cDNA was cloned from an Sf9 cDNA library. This cDNA encodes a 299-amino acid protein named Sf caspase-1 proenzyme (Sf procaspase-1) (Fig. 1), with a predicted molecular mass of ~35 kDa. Sequence alignment of Sf procaspase-1 with all known caspases revealed that it has highest homology to the human downstream apoptotic effectors Mch3 (42% identity) (24), CPP32 (38% identity) (25), and Mch2alpha (38% identity) (23), followed by other family members. Additionally, Sf procaspase-1 belongs to the Ced-3 subfamily (7, 8) which includes the proenzymes of Ced-3, CPP32, Mch2, Mch3, Mch4, Mch5, and Mch6 (7, 8). Sf procaspase-1 is also structurally similar to other caspases. A mature Sf caspase-1 could be derived from the precursor proenzyme by cleavage at Asp-195 to generate the two subunits, and Asp-15 and Asp-28 which would remove the prodomain. Interestingly, Sf caspase-1 has a QAC<UNL>Q</UNL>G active site pentapeptide, identical to that of Mch4 and Mch5 (7).


Fig. 1. Predicted amino acid sequence of Sf procaspase-1. The active site pentapeptide QACQG is boxed. Cleavage sites after Asp-28, Asp-184, and Asp-195 are indicated by vertical arrows. The N termini of the two subunits (p19/p18 and p12) are indicated by horizontal arrows.
[View Larger Version of this Image (24K GIF file)]


Expression, Purification, and Microsequencing of Sf Caspase-1

To determine the enzymatic activity and primary structure of Sf caspase-1 and the exact autocatalytic processing sites in its proenzyme, it was expressed in bacteria, purified, and microsequenced. This is because bacteria do not contain any caspase activity, and mutant Cys right-arrow Ala active site caspases are not autoprocessed in bacteria. Expression of Sf procaspase-1, containing N-terminal T7-tag and C-terminal His6-tag, produced soluble mature enzyme. As shown in Fig. 2A, purified mature Sf caspase-1 migrates in SDS-gels as three bands of apparent molecular masses of 19, 18, and 13 kDa. The N terminus of the 13-kDa band starts with Gly-196, indicating that processing occurred after Asp-195 of Sf procaspase-1. The calculated molecular mass of this peptide excluding the C-terminal His6-tag is ~12 kDa. The N termini of the 19-kDa and 18-kDa bands start with Ala-29, indicating that processing occurred after Asp-28 of Sf procaspase-1. Processing at these residues removes a 4-kDa prodomain. Site-directed mutagenesis of Asp-184 and Asp-195 revealed that the difference in size between the two polypeptides is due to processing at Asp-184 in the case of the 18-kDa polypeptide and Asp-195 in the case of the 19-kDa band (data not shown).


Fig. 2. Subunit structure of mature Sf caspase-1. Sf procaspase-1 was expressed in Escherichia coli, purified, analyzed by SDS-PAGE and Coomassie staining, and then microsequenced. A, lane M, molecular mass markers (kDa); lane 2, Ni2+-affinity-purified mature Sf caspase-1 enzyme. The N-terminal sequence of p12 is GSPSTSYRIPVHADFLIAFS. The N-terminal sequence of p19 and p18 is ALGSNSSSQPNRVARMPVDR. B, in vitro processing of Sf procaspase-1 by mature recombinant Sf caspase-1 enzyme. [35S]Methionine-labeled Sf procaspase-1 was incubated with pure mature Sf caspase-1 (1 ng/µl) for the indicated times (min) at 37 °C. The reaction products (2 µl/lane) were then analyzed by Tricine-SDS-PAGE and autoradiography. Full-length Sf procaspase-1 and the cleavage products are indicated.
[View Larger Version of this Image (22K GIF file)]


Similar results were also obtained after incubation of 35S-labeled Sf procaspase-1 with mature recombinant Sf caspase-1 (Fig. 2B). Sf caspase-1 was able to process its proenzyme in a time-dependent fashion to generate the p19, p18, and p12 species. Based on these data, Sf procaspase-1 can autoprocess after Asp-28, Asp-184, and Asp-195 to generate the two subunits (p19/p18, large subunit, and p12, small subunit) of mature Sf caspase-1 enzyme.

p35 Is a Substrate and a Potent Inhibitor of Sf Caspase-1

Sf9 insect cells respond to baculovirus infection by activating a novel caspase to initiate apoptosis (21). This process is counteracted by expression of the baculovirus-encoded protein p35 which is a substrate for, and a potent inhibitor of, members of the caspase family (19, 20).

To determine whether p35 is a substrate for Sf caspase-1, purified recombinant Sf caspase-1 was incubated with 35S-labeled p35 for various times (Fig. 3A). This generated the expected 10- and 25-kDa fragments, indicative of cleavage at Asp-87 (Fig. 3C). Identical results were also obtained with apoptotic extract from p35 null mutant virus-infected Sf9 cells (Fig. 3B, lane 2) and recombinant human CPP32 (lane 4) and Mch3 (lane 5).


Fig. 3. Cleavage of p35 by Sf caspase-1, CPP32, and Mch3. [35S]Methionine-labeled p35 was incubated with pure mature Sf caspase-1 (1 ng/µl) for the indicated times (min) at 37 °C (A) or buffer (lane 1), apoptotic extract from vp35Delta baculovirus-infected Sf9 cells (1 µg/µl) (lane 2), Sf caspase-1 (5 ng/µl) (lane 3), recombinant hCPP32 (3 ng/µl) (lane 4), or recombinant hMch3 (2 ng/µl) (lane 5) for 1 h at 37 °C (B). The reaction products were then analyzed by Tricine-SDS-PAGE and autoradiography. The full-length p35 and the 25-kDa and 10-kDa cleavage products are indicated. The 25-kDa product is less radioactive because of lower methionine content. C, a schematic diagram illustrating the site of cleavage and the expected cleavage products of baculovirus p35 after interaction with Sf caspase-1, CPP32, or Mch3.
[View Larger Version of this Image (25K GIF file)]


Sf caspase-1 was potently inhibited by purified recombinant p35 in a dose-dependent manner (IC50 ~0.5 nM) (Fig. 4). The endogenous protease activity in Sf9 apoptotic extract was also potently inhibited by p35 (IC50 ~0.5 nM) and exhibited a similar dose dependence. Interestingly, unlike recombinant Sf caspase-1, the protease activity in the Sf9 apoptotic extract was not completely inhibited by high concentrations of p35. This suggests that this extract contains an additional protease activity distinct from Sf caspase-1 which is not sensitive to p35. This activity could be another novel caspase that is activated by viral infection. The poxvirus CrmA, which is a potent inhibitor of ICE, had very little effect on the activity of recombinant Sf caspase-1 or Sf9 apoptotic extract under the same conditions (data not shown). These results clearly suggest that Sf caspase-1 could be an important target of baculovirus p35 during viral infection of S. frugiperda insect cells.


Fig. 4. Inhibition of Sf caspase-1 by recombinant p35. Equivalent protease activity of recombinant Sf caspase-1 (50 pg/µl) or apoptotic lysate from vp35Delta baculovirus-infected Sf9 cells (1 µg/µl) was incubated with the indicated concentrations of recombinant p35 and then assayed for protease activity using the tetrapeptide substrate DEVD-AMC (50 µM). The enzymatic activities were expressed as a percentage of protease activity in the absence of p35.
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The Nuclear Sf FKBP46 Is a Target of Sf Caspase-1 in Baculovirus-induced Apoptosis

Recently, we identified and cloned an Sf9 nuclear immunophilin named FKBP46 (22). FKBP46 contains two N-terminal acidic domains with uninterrupted stretches of polyglutamic/aspartic acid residues. Because of the high content of Asp residues in these domains, we decided to test whether FKBP46 is a target of Sf caspase-1 in apoptosis. Sf9 cells were infected with wild type (WT) baculovirus (encodes p35) or p35 null mutant (vp35Delta ) baculovirus and harvested 24 h after infection. Western blot analysis revealed that FKBP46 is cleaved to an ~25-kDa fragment in Sf9 cells infected with vp35Delta but not with wild type virus (Fig. 5, lanes 3 and 4, respectively). Since cells infected with vp35Delta virus but not wild type virus undergo rapid apoptosis as a result of activation of Sf caspase-1, it is most likely that Sf caspase-1 is the enzyme responsible for cleaving FKBP46. This was supported by our observations that incubation of recombinant Sf caspase-1 with purified recombinant FKBP46 or Sf9 nuclei yielded the same ~25-kDa cleavage product (lanes 2 and 5). Also, overexpression of Sf procaspase-1 in Sf9 cells resulted in its processing as determined by immunostaining with T7 antibody (Fig. 6A) and generation of maximal Sf caspase-1 activity at 46 h postinfection (Fig. 6B). The lower Sf caspase-1 activity observed at 16-24 h postinfection (Fig. 6B) might be due to p35 inhibition. In addition, the lower T7-immunostaining observed at 46 h postinfection is due to removal of the T7-tagged prodomain by the high Sf caspase-1 activity. Maximal cleavage of FKBP46 (Fig. 6C) and induction of apoptosis with characteristic internucleosomal DNA cleavage (Fig. 6D) were observed at 46 h postinfection. About 60% of cells infected with Sf caspase-1 baculovirus showed typical morphological changes of apoptosis-like blebbing and nuclear condensation at 46 h postinfection.


Fig. 5. Cleavage of the Sf nuclear immunophilin FKBP46 by Sf caspase-1 and during vp35Delta baculovirus-induced apoptosis. Purified recombinant Sf FKBP46 (20 ng/µl) (lanes 1 and 2) or nuclei from wild type virus-infected Sf9 cells (~0.5 µg/µl) (24 h postinfection, lanes 4 and 5) were incubated with (+) or without (-) recombinant Sf caspase-1 (5 ng/µl) for 2 h at 37 °C and then analyzed by SDS-PAGE and immunoblotting with an FKBP46-specific polyclonal antibody. Nuclei from vp35Delta baculovirus-infected Sf9 cells (Delta p35, lane 3) were isolated and directly analyzed by SDS-PAGE and immunoblotting. Full-length and 25-kDa cleavage products of FKBP46 are indicated.
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Fig. 6. Overexpression of Sf procaspase-1 in Sf9 cells results in its activation, cleavage of FKBP46, and DNA cleavage. Sf9 cells were infected with wild type baculovirus or recombinant baculovirus encoding T7-tagged Sf procaspase-1. At the indicated times (A, B, and C), the cells were harvested, fractionated into cytosolic and nuclear fractions, and 20 µg of each cytosolic fraction was analyzed by Western blotting with a T7-antibody (A) or by enzymatic assay with the DEVD-AMC (50 µM) peptide substrate (B). The nuclear fractions (20 µg/lane) were analyzed by Western blotting with the FKBP46 antibody (C). D, induction of DNA cleavage by overexpressed Sf caspase-1. Total DNA was isolated from Sf9 cells infected with wild type baculovirus (negative control, lane 1) or recombinant baculoviruses encoding ProICEgamma (positive control, lane 2) or Sf procaspase-1 (lane 3) and then analyzed by 1.8% agarose-gel electrophoresis.
[View Larger Version of this Image (38K GIF file)]


In conclusion, we have identified and characterized two novel components of the apoptotic machinery of the insect S. frugiperda, the host of the baculovirus A. californica. The death effector component is a caspase named Sf caspase-1, related to the mammalian apoptotic effectors Mch3, CPP32, and Mch2alpha . Mature Sf caspase-1 can cleave the baculovirus antiapoptotic protein p35, is potently inhibited by p35, and exhibits similar p35-inhibitory profile as the endogenous Sf9 protease present in Sf9 apoptotic extracts. Thus, Sf caspase-1 is most likely an important target of baculovirus p35. The second component is a death-associated substrate known as FKBP46, which is an Sf9 nuclear DNA binding immunophilin recently discovered in our laboratory. We demonstrated that FKBP46 is cleaved specifically during vp35Delta baculovirus-induced apoptosis of Sf9 cells and by the death effector component Sf caspase-1. Because the basic apoptosis program has been highly conserved during evolution, the identification of these two components should facilitate the efforts to elucidate the molecular mechanism and the physiological significance of apoptosis in diverse organisms ranging from insects to mammals.


FOOTNOTES

*   This work was supported by Research Grants AG 13487 and AI 35035 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBankTM/EMBL Data Bank with accession number(s) U81510[GenBank].


Dagger    The first two authors contributed equally to this work.
§   To whom correspondence should be addressed: Jefferson Medical College, Kimmel Cancer Institute, Bluemle Life Sciences Bldg., 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4632; Fax: 215-923-1098; E-mail: E_Alnemri{at}lac.jci.tju.edu.
1    The abbreviation used is: ICE, interleukin 1beta converting enzyme; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Tricine, N-tris(hydroxymethyl)methylglycine.

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