Biochimica et Biophysica Acta 1499 (2001) 191^198
www.elsevier.com/locate/bba
Cloning and characterization of an inhibitor of apoptosis protein (IAP)
from Bombyx mori
Qihong Huang
a;b
, Quinn L. Deveraux c , Susumu Maeda b , Henning R. Stennicke d ,
Bruce D. Hammock b , John C. Reed d; *
a
Microbiology Program, University of California, Davis, One Shields Ave., Davis, CA 95616, USA
Department of Entomology, University of California, Davis, One Shields Ave., Davis, CA 95616, USA
c
Genomics Institute of the Novartis Research Foundation, 3115 Merry¢eld Row, Suite 200, San Diego, CA 92121, USA
Program on Apoptosis and Cell Death Regulation, The Burnham Institute, 10901 N. Torrey Pines Rd, La Jolla, CA 92037, USA
b
d
Received 14 July 2000; received in revised form 5 September 2000; accepted 14 September 2000
Abstract
We cloned a novel inhibitor of apoptosis protein (IAP) family member, BmIAP, from Bombyx mori BmN cells. BmIAP
contains two baculoviral IAP repeat (BIR) domains followed by a RING domain. BmIAP shares striking amino acid
sequence similarity with lepidopteran IAPs, SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP,
suggesting evolutionary conservation. BmIAP blocks programmed cell death (apoptosis) in Spodoptera frugiperda Sf-21 cells
induced by p35 deficient Autographa californica nucleopolyhedrovirus (AcMNPV). This anti-apoptotic function requires
both the BIR domains and RING domain of BmIAP. In mammalian cells, BmIAP inhibits Bax induced but not Fas induced
apoptosis. Further biochemical data suggest that BmIAP is a specific inhibitor of mammalian caspase-9, an initiator caspase
in the mitochondria/cytochrome-c pathway, but not the downstream effector proteases, caspase-3 and caspase-7. These
results suggest that suppression of apoptosis by lepidopteran IAPs in insect cells may involve inhibition of an upstream
initiator caspase in the conserved mitochondria/cytochrome-c pathway for apoptosis. ß 2001 Elsevier Science B.V. All
rights reserved.
Keywords: Apoptosis; Insect; Inhibitor of apoptosis protein; Caspase; Cell death; Protease inhibitor
1. Introduction
Apoptosis or programmed cell death is a cellular
suicide process in which damaged or harmful cells
are eliminated from multicellular organisms [1]. Cells
undergoing apoptosis have distinct morphological
changes including cell shrinkage, membrane blebbing, chromatin condensation, apoptotic body for-
* Corresponding author. Fax: +1-858-646-3140;
E-mail: [email protected]
mation and fragmentation [1]. This cell suicide program is evolutionarily conserved across animal
species [2]. Apoptosis plays an important role in
the development and homeostasis of metazoans [3]
and is also critical in insect embryonic development
and metamorphosis [4,5]. Furthermore, apoptosis
acts as a host defense mechanism by which virally
infected cells are eliminated to limit the propagation
of viruses [6]. Dysregulation of apoptosis has been
associated with a variety of human diseases including
cancer, neurodegenerative disorders and autoimmune
diseases [7].
0167-4889 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 4 8 8 9 ( 0 0 ) 0 0 1 0 5 - 1
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Inhibitor of apoptosis proteins (IAPs) were originally discovered in baculoviruses [8,9]. CpIAP and
OpIAP are able to block apoptosis induced by p35
de¢cient baculovirus AcMNPV in insect Sf-21 cells
[8,9]. Cellular IAP homologues have been found in
various animal species including worms, insects and
humans [10,11]. IAP proteins have distinctive primary structure. They contain one to three copies of
`baculoviral IAP repeat' (BIR) domain and most
IAPs also contain a RING domain near their C-termini. The BIR domain contains a highly conserved
arrangement of Cys/His residues forming a stable
fold that chelates zinc [12,13]. The BIR region was
also found to interact with regulators of IAPs including Grim, Reaper and Hid from Drosophila and may
also mediate homo-oligomerization [14^20]. The
RING ¢nger is a common zinc binding motif that
also exists in other cellular proteins [21]. Recent studies show that several IAPs, including XIAP, cIAP1,
cIAP2, DIAP1, SfIAP and CpIAP, are inhibitors of
caspases, a family of intracellular proteases responsible for the execution of the apoptosis program [22^
28]. These IAPs can directly bind and inhibit some
members of caspase family including caspase-3, -7
and -9. Structure^function studies have demonstrated that inhibition of caspase-3 and -7 requires
only a single BIR domain while the RING domain
may perform other functions including recruitment
of ubiquitin conjugating enzymes [29]. When expressed without the associated BIRs, the RING region of SfIAP was found to enhance the proapoptotic activity of mammalian caspase-9 suggesting
this domain operates as a trans-dominant inhibitor
of endogenous proteins involved in apoptosis suppression [28]. Ectopic expression of lepidopteran
SfIAP and baculoviral CpIAP blocks apoptosis in
mammalian cells, suggesting conservation of the apoptosis program among various species and a shared
mechanism used by the IAP family [28].
Bombyx mori (silkworm) has been domesticated
for silk production for thousands of years. It has
also been developed as an organism for large scale
production of foreign proteins in the biotechnology
industry [30]. Despite its extensive use in sericulture
and biotechnology, no apoptosis regulating genes of
silkworm have been identi¢ed so far. Here we report
the cDNA cloning and characterization of a cellular
IAP from a cell line (BmN) derived from B. mori and
demonstrate that BmIAP is a direct inhibitor of
mammalian caspase-9.
2. Materials and methods
2.1. Cloning of BmIAP
mRNA was isolated from BmN cells by using a kit
from Qiagen. Degenerate primers were designed
according to the consensus amino acid sequences
between baculoviral IAPs and Drosophila IAPs:
5P-GC(A/C/G/T)GA(A/C/G/T)GC(A/C/G/T)GG(A/
C/G/T)TT(T/C)TT(T/C)TA-3P and 5P-AC(A/C/G/T)AC(A/G)TG(A/C/G/T)CC(A/G)CA(A/C/G/T)GG3P. Reverse transcription-polymerase chain reaction
(PCR) was performed for 35 cycles by using 94³C
for 45 s, 46³C for 1 min and 72³C for 1 min. Ampli¢ed fragments were blunt-end cloned into the
HincII site of pTZ-19 and then sequenced. To obtain
full length BmIAP cDNAs, 5P rapid ampli¢cation of
cDNA ends (RACE) and 3P RACE were performed
by using commercial kits (Gibco BRL; Takara) and
5P-CTGTTCCCACGGAACGTC-3P and 5P-GCCACCAATGATTCGAC-3P as internal PCR primers.
2.2. Plasmid construction
A cDNA fragment encompassing the complete
open reading frame (ORF) of BmIAP was PCR ampli¢ed and subcloned into the EcoRI^XhoI sites in
pcDNA3-myc and pGEX4T-1. Plasmids encoding
fragments of BmIAP, including BIR1+2 (amino
acid residue 1^292) and RING (residue 293^346),
were ampli¢ed by PCR by using primers containing
either start or stop codons as appropriate and subcloned into pTZ-19 and pcDNA3-myc plasmids.
2.3. Protein expression and puri¢cation
pGEX4T-1-BmIAP plasmid was introduced into
Escherichia coli strain BL21 (DE3) containing the
plasmid pT-Trx. Glutathione S-transferase (GST) fusion proteins were obtained by induction with 0.05
mM isopropyl L-thiogalactoside at 25³C for 8 h and
then puri¢ed using glutathione-Sepharose [28]. The
catalytic domains of caspase-3, caspase-7 and caspase-9 were expressed, puri¢ed by Ni-chelation a¤n-
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ity chromatography, and quanti¢ed as described [31^
33].
2.4. Cell extracts and caspase assays
Cytosolic extracts were prepared by using human
embryonic kidney (HEK) 293 cells [22]. For initiating
caspase activation, 1 WM horse heart cytochrome-c
(Sigma) and 1 mM dATP was added to extracts [24].
Caspase activity was assayed by release of 7-amino4-tri£uoromethyl-coumarin (AFC) from Ac-DEVDAFC or Ac-LEHD-AFC (Calbiochem), using a spectro£uorimeter as described [31,34].
2.5. Cell culture, transfection and apoptosis assays
Insect Sf-21 cells were maintained at 27³C in Excell 401 medium (JRH Biosciences, Lenexa, KS,
193
USA) supplemented with 2.5% fetal bovine serum
(FBS). vP35del, AcMNPV containing a deletion in
the p35 gene, was propagated in TN-386 cells [35].
Plasmids encoding full length or deletion mutants of
BmIAP (1 Wg) were co-transfected with 1 Wg vP35del
viral DNA into Sf-21 cells by using lipofectin from
Gibco BRL. Occlusion body formation was observed
under light microscopy 3 days post-transfection.
HEK293 cells were maintained in Dulbecco's
modi¢ed Eagle's medium (Irvine Scienti¢c) supplemented with 10% FBS, 1 mM L-glutamine and antibiotics. 293 cells (106 ) were cotransfected by using
Superfect (Qiagen) with 0.1 Wg of green £uorescence
protein (GFP) marker plasmid pEGFP (CLONTECH), 0.25 Wg of either pcDNA3-Bax or
pcDNA3-Fas and 1.5 Wg of pcDNA3-myc-BmIAP.
Both £oating and adherent cells were recovered 24^
36 h post-transfection and pooled, and the percent-
Fig. 1. Predicted domain topology and amino acid sequence of BmIAP. (A) The BIR and RING domains of BmIAP are depicted.
(B) The predicted amino acid sequence of BmIAP is presented. Sequence alignments of the BIR1 (C), BIR2 (D) and RING (E) domains of BmIAP with the corresponding domains of other IAP family members are shown. Bold text indicates identical amino acid.
The GenBank accession numbers of sequences used for the alignments are: B. mori IAP (BmIAP) AF281073, Spodoptera frugiperda
IAP (SfIAP) AF186378, Trichoplusia ni IAP (TnIAP) AF195528, Orgyia pseudotsugata nucleopolyhedrovirus IAP (OpIAP) P41437,
Cydia pomonella granulovirus IAP (CpIAP) P41436, and D. melanogaster IAP1 (DIAP1) Q24306.
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age of GFP-positive cells with nuclear apoptotic
morphology was determined by staining with
0.1 Wg/ml 4P-6-diamidino-2-phenylindole (DAPI)
(mean þ S.D.; n = 3) [36].
3. Results
3.1. Cloning of BmIAP
The full length BmIAP cDNA (GenBank accession
number AF281073) contains a continuous ORF encoding a protein of 346 amino acids (Fig. 1). This
ORF is initiated by an AUG within a favorable context for translation [37] and is preceded by upstream
stop codons in all three reading frames. Similar to
SfIAP, the predicted BmIAP protein contains two
BIR domains followed by a RING domain near its
C-terminus (Fig. 1A,B). In the BIR domain of
BmIAP, the conserved presence and spacing of cysteine and histidine residue (CX2 CX6 WX9 HX6 C) is
also observed. Within BIR and RING regions,
BmIAP shares 88% amino acid identity (92% similarity) with SfIAP, 90% identity (92% similarity) with
TnIAP and 76% identity (81% similarity) with
CpIAP (Fig. 1C^E). Thus BmIAP shares high sequence similarity with the other two lepidopteran
IAPs, SfIAP and TnIAP, suggesting evolutionary
conservation.
3.2. Both BIR and RING domains of BmIAP are
required to block apoptosis induced by p35
de¢cient AcMNPV
To test the anti-apoptotic activity of BmIAP in
insect cells, we co-transfected AcMNPV p35 de¢cient
viral DNA into Sf-21 cells with plasmids containing
full length BmIAP or truncation mutants of BmIAP
lacking two BIR domains or the RING domain.
IAPs that have anti-apoptotic function in insect cells
are able to rescue p35 de¢cient AcMNPV, allowing
virus replication and resulting in occlusion body formation which serves as a visual screening end-point
under light microscopy [8,9]. Since SfIAP has been
shown to block apoptosis in both insect and mammalian cells [28], whereas AcIAP is ine¡ective [6,28],
SfIAP and AcIAP were used as positive and negative
controls, respectively (Fig. 2D,E).
Fig. 2. Rescue of p35 de¢cient AcMNPV by BmIAP. Sf-21 cells
were transfected with p35 de¢cient AcMNPV viral DNA and
plasmids encoding various IAPs or IAP fragments. Cells expressing IAPs with anti-apoptotic activity support virus replication whereas cells without a functional IAP are unable to support virus replication and undergo apoptosis. Production of
viable viral progeny from rescued viruses results in the formation of occlusion bodies, serving as a convenient end-point.
Note that Sf-21 cells co-transfected with p35 de¢cient AcMNPV
viral DNA and (A) BmIAP or (D) SfIAP show the production
of occlusion bodies as indicated by arrowheads, whereas (B)
BmIAP-BIR, (C) BmIAP-RING and (E) AcIAP show apoptotic
body formation (indicated by arrowheads) without occlusion
body formation. (F) Control uninfected SF-21 cells are shown
for comparison. Cells were photographed 3 days post-transfection at U40 magni¢cation.
A plasmid encoding the full length BmIAP was
able to complement the p35 de¢ciency in the baculovirus, supporting occlusion body production and virus replication at 3 days post-transfection (Fig. 2A).
In contrast, neither the BIR nor RING domain
deletion mutants of BmIAP were able to support
occlusion body formation. Sf-21 cells displayed morphological changes of apoptosis such as apoptotic
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Fig. 3. BmIAP protects mammalian cells against Bax induced
but not Fas induced apoptosis. Expression plasmid encoding
Bax (A) or Fas (B) were co-transfected into HEK 293 cells
with the indicated myc-tagged IAP expression plasmids. Percentage apoptosis was measured 24^36 h post-transfection by
DAPI staining (mean þ S.D., n = 3). (C) Recombinant BmIAP
(2 WM) was added to cytosolic extracts (10 mg/ml) from
HEK293 cells concurrently with the addition of 1 WM cytochrome-c/1 mM dATP. After incubation at 30³C for 10 min,
aliquots were withdrawn and assayed for caspase activity as
measured by release of AFC from Ac-DEVD-AFC substrate
(100 WM). Data are presented as a percentage relative to control reaction in which cytochrome-c/dATP were added alone.
195
indicated that the levels of the BIR and RING truncation proteins were similar to that of full length
BmIAP in transfected cells, excluding di¡erences in
protein levels as an explanation for the failure of the
BIR domains or RING domain to suppress cell
death (data not shown).
These results were further con¢rmed in a cell-free
system in which exogenously added cytochrome-c, an
agonist of the caspase-9 activating protein Apaf-1
[41], induces activation of caspase-3 and similar effector proteases, as measured by hydrolysis of AcDEVD-AFC [34]. In cytosolic extracts treated with
cytochrome-c, recombinant BmIAP and positive control recombinant SfIAP completely blocked the hydrolysis of Ac-DEVD-AFC whereas negative control
recombinant XIAP-BIR1 had no e¡ect on caspase
activity (Fig. 3C). Since caspase-3 and -7 are common to both Bax and Fas pathways, these results
suggest BmIAP, like SfIAP, inhibits the mitochondria/cytochrome-c pathway in mammalian cells,
thus, suppressing apoptosis at a step upstream of
caspase-3 and -7. This point is supported by the ob-
body formation when either the BIR or RING domain deletion mutants were co-transfected with p35
de¢cient viral DNA (Fig. 2B,C). These results demonstrate that both the BIR and RING domain regions of BmIAP are required in combination for the
anti-apoptotic function of BmNPV in insect cells.
3.3. BmIAP inhibits Bax induced but not Fas induced
apoptosis in mammalian cells
SfIAP and baculoviral IAPs were previously
shown to block apoptosis in mammalian cells
[28,38^40]. To explore whether BmIAP has similar
properties, we co-expressed BmIAP in HEK293 cells
with either Fas or Bax, representing two major pathways that utilize caspase-8 and caspase-9, respectively, as their apical proteases. Similar to SfIAP,
full length BmIAP inhibited Bax (Fig. 3A) but not
Fas induced apoptosis (Fig. 3B). In contrast human
XIAP protected cells against both Bax and Fas induced apoptosis. As in Sf-21 cells, the inhibition of
Bax induced apoptosis in mammalian cells also requires both the BIR and RING domains of BmIAP
suggesting the conservation of the structural requirements for inhibition (Fig. 3A). Immunoblot analysis
Fig. 4. BmIAP directly suppresses caspase-9 but not caspase-3
or caspase-7. Recombinant active (A) caspase-9 was added at
0.2 WM and incubated at 37³C with Ac-LEHD-AFC substrate
(100 WM) in the presence or absence of various concentration
(0.2^1.6 WM) of recombinant puri¢ed BmIAP or SfIAP. AFC
release was measured continuously. Data are expressed as a
percentage relative to control reactions lacking IAPs, using
rates determined from the linear portion of enzyme progress
curves. Various control GST-fusion proteins had no inhibition
e¡ect (not shown). (B) Recombinant caspase-3 (2.6 nM) was incubated at 37³C with Ac-DEVD-AFC substrate (100 WM) in
the presence or absence of 0.05 WM GST-XIAP, 0.5 WM GSTBmIAP (200-fold molar excess to caspase) or 0.5 WM GSTSfIAP (200-fold molar excess). AFC release was measured as
above. (C) Recombinant caspase-7 (7.0 nM) was incubated at
37³C with Ac-DEVD-AFC substrate (100 WM) in the presence
or absence of 0.14 WM GST-XIAP, 0.7 WM GST-BmIAP (100fold molar excess relative to caspase) or 0.7 WM GST-SfIAP
(100-fold molar excess). AFC release was measured as above.
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servation that BmIAP does not inhibit caspase-3 and
-7 in vitro (see Section 3.4).
3.4. BmIAP is a direct inhibitor of caspase-9
Since caspase-3, -7 and -9 are involved in the
apoptotic pathway induced by Bax and since SfIAP
has been shown to directly inhibit caspase-9 [28], we
examined the possibility that BmIAP is also a direct
caspase-9 inhibitor. Puri¢ed recombinant BmIAP
was incubated with puri¢ed recombinant caspase-9.
Residual activity was measured using Ac-LEHDAFC as a substrate of caspase-9. BmIAP inhibited
recombinant caspase-9 in a concentration dependent
manner. The relative amount of BmIAP required for
caspase-9 inhibition was Veight-fold molar excess,
similar to the results reported previous for SfIAP
and XIAP (Fig. 4A) [26,28]. Unlike XIAP, but similar to SfIAP, BmIAP did not inhibit recombinant
caspase-3 and caspase-7, suggesting a narrower speci¢city compared to human XIAP (Fig. 4B,C).
4. Discussion
Studies of lepidopteran SfIAP and baculoviral
CpIAP demonstrate that they block apoptosis by inhibiting caspases [28]. In this report, we cloned a
novel lepidopteran IAP, BmIAP, from B. mori and
provide direct evidence that this lepidopteran IAP
can also function as a caspase inhibitor. BmIAP
shares strong amino acid sequence similarity with
SfIAP, TnIAP and baculoviral CpIAP. Structure^
function analysis of BmIAP reveals identical domain
requirements (e.g. BIR plus RING) for suppression
of apoptosis in both insect and mammalian cells and
similar caspase selectivity (e.g. caspase-9) compared
to the previously characterized lepidopteran and baculovirus IAPs. These results strongly support the
idea that lepidopteran IAPs are evolutionary conserved in both sequence and function.
Similar to SfIAP and CpIAP, ectopic expression of
BmIAP blocks Bax induced but not Fas induced
apoptosis in mammalian cells. This inhibitory e¡ect
requires both the BIR and RING domains. Since
caspase-3 and -7 operate at the point of convergence
of the Bax and Fas pathways, these observations
suggested that caspase-9 is the speci¢c target of
BmIAP. Direct con¢rmation of inhibition of caspase-9 by BmIAP was obtained by in vitro studies,
revealing that puri¢ed recombinant BmIAP directly
inhibits mammalian caspase-9 in vitro. The interesting question remains what is the endogenous caspase
target of BmIAP in silkworm. Baculoviral IAPs
which share a high degree of amino acid sequence
identity with BmIAP have been shown to prevent
activation of Sf-procaspase-1, a putative counterpart
of mammalian downstream caspases, but do not directly inhibit active Sf-caspase-1 [42]. We therefore
speculate that the target of BmIAP is an upstream
insect caspase which is analogous to caspase-9. The
recently published genome from Drosophila melanogaster identi¢ed eight caspases [43]. Three of these
caspases contain long prodomains and thus are likely
to serve as candidates of upstream initiator caspases
analogous to caspase-9 of mammals. A recent study
also showed that DIAP1, a counterpart of BmIAP in
£ies, directly binds and inhibits DRONC, one of the
three putative upstream insect caspases [19]. Cloning
and characterization of caspases from B. mori will
shed new light on the speci¢c target of BmIAP and
yield a better understanding of apoptotic pathways
in B. mori.
Drosophila and mammalian IAPs have been shown
to play important roles in the development of these
organisms [44,45]. By analogy, it seems reasonable to
speculate that BmIAP is also a critical player in silkworm development. We therefore envision that manipulation of apoptosis during the development of
the silkworm could have a positive impact on the
sericulture and biotechnology industries in the future.
Acknowledgements
We would like to thank Dr. Lois K. Miller for the
vP35del virus. This work was supported by grants
from the National Institute of Health (ES02701,
AG15402, ES 04699), the US Department of Agriculture (97-35302-4406, 9802852), Binational Agriculture Research and Development (96-34339-3532),
IDUN Pharmaceuticals, Center grants from the National Institute of Environmental Health Science (ES
05707) and National Cancer Institute (CA30199) and
a fellowship from the Leukemia Society of America
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Q. Huang et al. / Biochimica et Biophysica Acta 1499 (2001) 191^198
(Q.L.D). We dedicate this paper to Professor Susumu Maeda, who passed away during the completion
of the project.
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