From the
Interleukin-1
ICE
There is increasing evidence for
an involvement of other ICE family members in apoptotic pathways. For
example, a family member named Nedd-2 in mouse and ICH-1 in humans
induced apoptosis when overexpressed in mammalian
cells(9, 10) . Similarly, overexpression of CPP32,
another ICE family member which was isolated from human cells, induced
apoptosis in Sf9 insect cells(11) . Finally, another link
between ICE family members and apoptosis was recently suggested by
experiments showing that poly(ADP-ribose) polymerase, a protein that is
processed in cells undergoing apoptosis, may be cleaved by an ICE-like
chicken protease called prICE(12, 13) . Although ICE
itself may participate directly in the diverse biological processes of
cytokine maturation and apoptosis, it is also possible that in
vivo, a related enzyme (or enzymes) plays a more pivotal role in
one or both of these pathways.
Recently we generated ICE-deficient
mice and observed that these mice are deficient in the production of
mature IL-1
The P1 clones
denoted DMPC-HFF#1-319-D4 and DMPC-HFF#1-618-B9 were
isolated from the DuPont Merck Pharmaceutical Co. human foreskin
fibroblast P1 library 1 by Genome Systems Inc. The following PCR
primers derived from the human ICE coding sequence were used for the
isolation of these clones: GACATGACTACAGAGCTGG and ACCACGGCAGGCCTGGAT.
To generate the recombinant baculovirus, the transfer
vectors described above were used to cotransfect Sf9 cells with
modified and linearized AcMNPV DNA using the BaculoGold
The resultant plasmid,
pMCH-1/N-His ICH-2 32 kDa, was transformed into E. coli strain
MM294 (F-endA1 hsdR17 (r
Two bacteriophage P1 clones containing ICE sequences were
analyzed using PCR and hybridization mapping. One of these clones also
contains ICH-2 sequences, suggesting that ICH-2 is
located in the vicinity of the ICE gene on human chromosome 11 band
q22.2-q22.3 (data not shown)(19) .
ICE has been shown to cleave human pro-IL-1
We have identified a new
member of the ICE cysteine protease family, a family with implicated or
proven roles in both inflammation and apoptosis. The high degree of
sequence conservation between ICE and ICH-2 suggests that the two
proteases have similar or overlapping functions in vivo.
However, it is clear that ICE and ICH-2 are not entirely redundant.
ICH-2 is expressed in mice,
Activity
assays were performed in triplicate in the presence of inhibitors at
the indicated concentrations as described under ``Materials and
Methods.'' Results are expressed as percent of control (no
inhibitor) reactions.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
converting enzyme (ICE) is a cytoplasmic
cysteine protease required for generating the bioactive form of the
interleukin-1
cytokine from its inactive precursor. We report the
identification of ICH-2, a novel human gene encoding a member
of the ICE cysteine protease family, and characterization of its
protein product. ICH-2 mRNA is widely expressed in human
tissues in a pattern similar to, but distinct from, that of ICE.
Overexpression of ICH-2 in insect cells induces apoptosis. Purified
ICH-2 is functional as a protease in vitro. A comparison of
the inhibitor profiles and substrate cleavage by ICH-2 and ICE shows
that the enzymes share catalytic properties but may differ in substrate
specificities, suggesting that the two enzymes have different functions in vivo.
(
)is a member of a growing family of
proteins involved in both cytokine maturation and apoptosis. ICE is an
intracellular cysteine protease required for the proteolysis of the
inflammatory cytokine interleukin-1
(IL-1
) to its
biologically active form(1, 2, 3, 4) .
Unexpectedly, a role for ICE in programmed cell death, or apoptosis,
was suggested by the cloning of ced-3, an invertebrate gene
whose product is required for apoptosis during development in Caenorhabditis elegans, and the observation that the Ced-3
protein is 29% identical with ICE(5) . A role for ICE in
apoptotic pathways was further supported by experiments showing that
overexpression of ICE in a rat fibroblast cell line caused apoptosis,
and this activity could be blocked by CrmA, a cowpox virus protein that
is a selective inhibitor of ICE function(6) . Moreover,
introduction of CrmA protein into chicken dorsal root ganglion cells or
murine mammary epithelial cells blocked apoptosis induced by nerve
growth factor deprivation or by the absence of extracellular matrix,
respectively(7, 8) .
, but develop normally (3). Thymocytes and macrophages
from these animals undergo normal apoptosis with a variety of
stimulatory signals. This suggests that despite its ability to induce
apoptosis in vitro, ICE is not absolutely required for
apoptosis in these murine cell types. To identify novel ICE-related
proteins that may function in apoptosis or other biological processes,
we have begun to clone human genes with sequence homology to human ICE
(hICE). We report here the discovery of one such gene, ICH (ICE and Ced-3 homolog)-2, and a preliminary characterization of
its protein product. This is the first demonstration and
characterization of the protease activity of an ICE-related protein.
Cloning of ICH-2
An oligo(dT) and random-primed
human thymus cDNA library (Clontech) was screened with a 1,241-bp probe
containing the entire human ICE coding sequence. The probe was
generated by cleaving ICE5-1, a pGEM (Promega)-derived plasmid
containing the human ICE coding sequence, with XhoI and BamHI. Hybridization of 4 10
plaque-forming units was performed overnight at 55 °C in 10%
dextran sulfate, 0.1% sodium dodecyl sulfate (SDS), 1.25
Denhardt's solution, 5
SSC, 500 ng/ml poly(A), and 50
µg/ml sheared salmon sperm DNA. Filters were washed in 2
SSC, 0.1% SDS at 50 °C. Forty-three positive plaques were isolated,
and phage inserts were sequenced. Five clones contained inserts with
overlapping segments of the ICH-2 gene. One phage isolate
(Th18-3) contained an approximately 1.5-kb insert in which there was a
1,131 bp open reading frame. Both strands of this phage insert were
completely sequenced using an ABI automated sequencer.
Northern Blot Analysis
Human adult tissue Northern
blot membranes were purchased from Clontech. Each lane contains 2
µg of pure poly(A) RNA. The ICH-2-specific probe was a 264-bp fragment consisting of bases
1-250 of the ICH-2 coding sequence plus a 14-bp tail
introduced by the PCR reaction. PCR primers used to generate this
fragment were CCCACTAGTTCCCTATGGCAGAAGGCAACCA and GGGATATTTGGTCTATGTT.
The hICE-specific probe was a 347-bp fragment derived from bases 1 to
347 of the hICE coding sequence. The sequences of the PCR primers used
to generate this fragment were CCCCTCGAGGCCATGGCCGACAAGGTCCTGAAGGAG and
GGAAGAAAGTACTCCTTGAGAG. The human
-actin control probe was
supplied with the Northern blots (Clontech). Hybridization with the ICH-2 probe (25 ng of DNA, specific activity: 7
10
cpm/µg) was performed overnight at 65 °C in 5
SSPE (0.9 M NaCl, 0.5 M Na
PO
, 0.005 M EDTA, pH 7.7), 10
Denhardt's solution, 100 µg/ml sheared salmon sperm
DNA, 50% formamide, 2% SDS and was followed by two washes at 60 °C
in 2
SSC, 0.1% SDS. Hybridization with the ICE probe (50 ng of
DNA, specific activity: 5
10
cpm/µg) was
performed as above except the annealing temperature was 60 °C and
the wash temperature was 55 °C.
Generation of Recombinant
Baculoviruses
Recombinant transfer vectors were constructed by
subcloning of PCR generated ICE and ICH-2 cDNAs into the BamHI and NotI cut baculovirus transfer vector
pVL1393 (Invitrogen). The insert fragments containing the ICE and ICH-2 cDNAs were generated by PCR with a human ICE cDNA
plasmid (14) or with ICH-2 phage clone Th18-3 (see
above) as the respective templates. The 5` PCR primers for ICE were:
GCCGGGATCCTATAAATATGGCCGACAAGGTCCTGAAGGAG (p45),
CGGGATCCTATAAATATGCACCACCATCATCACCACGGATCTGGTCATATTGATGATGATGATAAGAACCCAGCTATGCCCACATCCTCAGGC
(p32), GCCGGGATCCTATAAAATGAACCCAGCTATGCCCACATCCTCAGGC (P20). The 3`
primers were CTGCGCGGCCGCATTTTAATGTCCTGGGAAGAGGTAG (p45 and p32),
CTGCGCGGCCGCTTAATCTTTAAACCACACCACACCAGG (p20). The 5` PCR primers for
ICH-2 were:
CGGGATCC-TATAAATATGCACCACCATCATCACCACGGATCTGGTCATAT-TGATGATGATGATAAGGCAGAAGGCAACCACAGAAAAAAG
(p44) and
CGGGATCCTATAAATATGCACCACCATCATCACCACGGATCT-GGTCATATTGATGATGATGATAAGGCCCTCAAGCTTTGTCCTCAT
(p30), and the 3` primer for both p44 and p30 was
ATAGTTTAGCGGCCGCAATTTCAATTGCCAGGAAAGAGGTAG. These primers introduced
restriction sites for subcloning as well as an NH-terminal
polyhistidine sequence tag to p32 ICE and both forms of ICH-2. The
correct clones were confirmed by restriction enzyme digestion and DNA
sequencing.
transfection system (PharMingen). The cell culture supernatants
containing baculovirus were harvested four days posttransfection and
were plated for single plaques. Recombinant viral plaques were visually
identified after neutral red staining (0.375 mg neutral red/ml of
plating top agar) and PCR (primers GGATTATTCATACCGTCCCACCATC and
GTGAGTTTTTGGTTCTTGCCGGGTCC) was performed on 10 µl of 100-µl
single plaque suspensions in serum-free Grace's medium (Life
Technologies, Inc.) to confirm insert size. After two rounds of
replating, the purified recombinant viruses were used to generate high
titer stocks. Sf9 cell infections were conducted at a multiplicity of
infection of 5 in 60-mm dishes containing 3
10
cells, and DNA was analyzed at 48 h postinfection.
Expression of ICH-2 in Escherichia coli
DNA
sequences encoding amino acids 105-377 of ICH-2 were
PCR-subcloned into the unique EcoRI restriction site of the E. coli expression vector pMCH-1 using the following primers:
GGGGAATTCATGGGTCATCATCATCATCATCA-TGGTAGCGGTCATATCGACGACGACGACAAGGCTCTGAAACTGT-GTCCGCATGAAGAGTTCCTGAGACTATG
and GGGGGATCCTCTATTAATTGCCAGGAAAGAGGTAG. This subcloning placed the ICH-2 gene under the control of the inducible bacteriophage
pL promoter and introduced an NH
-terminal
polyhistidine tag (six histidines) for purification on a
nickel-chelating column(15) . An enterokinase cleavage site was
also included to allow removal of the polyhistidine sequence.
Amplification with these PCR primers resulted in the modification of
the NH
-terminal coding sequences of the 32-kDa encoding
fragment to reflect E. coli codon usage. A silent mutation was
also added near the NH
terminus for removal of an EcoRI site to facilitate cloning.
-m
+) supE44 Thi-1)(16) which also contained a plasmid encoding the cI-857
temperature-sensitive pL promoter repressor. Growth of the transformed
strain at 40 °C as described below caused induction of the pL
promoter and expression of the 32-kDa N-His ICH-2 protein. An uninduced
overnight culture (grown at 28 °C) of the transformed bacterial
strain described above was used to inoculate (1:50) four 1-liter
cultures. These were grown at 28 °C to an A
value of 0.585 and then shifted to 40 °C. Cells were
harvested and frozen 1 h after induction, a time point that in a small
scale experiment gave a maximum of soluble protease activity (results
not shown).
Purification of ICH-2
Cell pellets were
resuspended in 100 ml of ice-cold lysis buffer (50 mM HEPES,
pH 7.5, 100 mM NaCl, 10% glycerol, 0.1% (w/v) CHAPS, 200
mM oxidized glutathione, 1 mM PMSF, 50 µM leupeptin, 1 µM pepstatin A). Cells were lysed in a
microfluidizer and centrifuged at 11,500 g for 30 min
at 4 °C. The lysate was diluted 1:1 with Buffer A (50 mM HEPES, 0.1 M NaCl, 10% (v/v) glycerol, pH 7.5) and passed
over a 1-ml Hi-Trap chelating Sepharose column (Pharmacia Biotech Inc.)
previously charged with 1 ml of 100 mM NiCl
,
washed with water, and equilibrated in Buffer A. The protein was eluted
with a gradient to Buffer B (Buffer A plus 0.5 M imidazole).
Fractions active in a protease assay (described below) were pooled and
stored at -80 °C. Expression and purification of N-His ICE
was analogous to that of N-His ICH-2, except that the purified protein
was dialyzed against buffer A to remove excess imidazole before
storage. The concentrations of N-His ICE and N-His ICH-2 were measured
by Coomassie Plus Protein Assay (Pierce), the latter after desalting a
sample on a Superdex 75 (Pharmacia) column.
Generation of Labeled Cleavage Substrates
PCR
primers 1 and 4 (see below) derived from the published human
pro-IL-1 sequence (17) were used to isolate the full-length coding
sequence in one step for subcloning into the XhoI and BamHI restriction sites of a T7 promoter containing plasmid
derived from pSV
(Clontech). The pro-IL-1
Asn
-Val
mutant was generated in a
three-step PCR reaction. First primers 1 and 3 were used to generate
the 5` mutant fragment, and primers 2 and 4 were used to generate the
3` mutant gene product. Second, the products of these reactions were
combined, and overlapping extension PCR was performed to generate the
assembled mutant product. Finally, the full-length mutant cDNA was
amplified with primers 1 and 4 for subcloning into the vector described
above. All PCR-generated fragments were sequenced after subcloning. PCR
primers used were as follows: 1) CCCCTCGAGTCTGAAGCAGCCATGGCAGAAGTACCT,
(2) GATAACCAGGCTTATGTGCACAACGTCCCTGTACGATC ACTGAACTGC, (3)
GCAGTTCAGTGATCGTACAGGGACGTTGTGCACATAAGCCA CGTTATC, (4)
CCCGGATCCGTACAGCTCTCTTTAGGAAGACACAAA (underlined bases indicate those
mutated to introduce the amino acid changes).
[
S]Methionine-labeled protein substrates for in vitro cleavage assays were generated using a coupled in
vitro transcription and translation system. 1 µg of plasmid
DNA was added directly to TnT
-coupled reticulocyte lysate
(Promega). Reactions were allowed to proceed for 60 min at 30 °C,
and the generated proteins were used immediately or stored at -20
°C. Cleavage assays were carried out in a reaction buffer of 100
mM HEPES, pH 7.0, 20% (v/v) glycerol, 5 mM dithiothreitol, 0.5 mM EDTA.
Enzymatic Activity Assays
Protease assays were
conducted in a reaction buffer of 100 mM HEPES, pH 7.0, 20%
(v/v) glycerol, 5 mM dithiothreitol, 0.5 mM EDTA.
Enzyme samples of 0.5-1.0 µg of protein were diluted to 80
µl with reaction buffer and inhibitors (when present) and
preincubated at 30 °C for 30 min. Reactions were initiated by
addition of 20 µl of reaction buffer containing the substrate
acetyl-Tyr-Val-Ala-Asp-p-nitroanilide (Ref. 18, Quality
Controlled Biochemicals, Inc., Hopkinton, MA). Incubations were
continued at 30 °C, and enzyme-catalyzed release of p-nitroanilide was monitored at 405 nm for 30 min in a
microtiter plate reader (Molecular Devices). To estimate K values, substrate concentration was
varied between 10 and 450 µM for N-His ICE and between 100
and 3,000 µM for N-His ICH-2. Substrate concentration was
500 µM for inhibitor studies, except as noted. All assays
were performed in triplicate.
Cloning of ICH-2
A human thymus cDNA library was
screened under low stringency hybridization conditions with a human ICE
gene probe. Of the 43 phage clones isolated, 5 contained ICH-2 sequences. The longest of these phage inserts contained a 1,131-bp
open reading frame that was highly homologous to human ICE (Fig. 1A). Although ICE and ICH-2 are less homologous
over the first 300 bases of their coding sequences (47%), they are very
related in the regions encoding the mature form of ICE (67%). Analyses
of genomic ICH-2 clones isolated in a similar screen of a
human genomic library allowed us to determine the intron/exon structure
of ICH-2. These data show that all of the intron positions are
conserved between ICE (19) and ICH-2 with the exception
of the second intron of ICE. This intron is absent in ICH-2,
and the low level of sequence conservation between the second exon of ICH-2 and the corresponding region of the ICE gene suggests
that the amino-terminal portions of these two genes may actually have
different evolutionary ancestry. To demonstrate that the gene we have
cloned is truly an expressed human gene, we performed reverse
transcription-PCR on human thymus mRNA with ICH-2-specific
primers. An approximately 1.1-kb product was generated, and its
identity was confirmed by sequencing (data not shown).
Figure 1:
Nucleotide sequence of ICH-2 and comparison of its predicted amino acid sequence with other ICE
family members. A, DNA coding sequence of ICH-2 and
its predicted amino acid sequence. The active site cysteine residue is boxed. Intron positions are indicated by arrows. B, protein sequence alignment of all human ICE family members
and of the mouse and rat ICE sequences. Dotted lines indicate
gaps introduced to allow optimal alignment of the sequences. Amino
acids are numbered to the right of each sequence. The
highly conserved pentapeptide containing the active site Cys is
indicated in bold type. The catalytic Cys and His residues are
indicated in bold type and are marked by an asterisk. The residues whose amino acid side chains form the P1 pocket are
indicated in bold type and are marked with a bullet (4, 9-11, 23, 28).
Mature ICE is
generated from its 404-amino acid precursor protein by removal of the
NH-terminal 119 amino acid ``prodomain'' and of
internal residues 298-316(4) , and so contains two
subunits called p20 and p10. The crystal structure of mature human ICE
complexed with peptidic inhibitors has been reported by ourselves and
another group(14, 20) . These structures show that ICE
is a tetramer of two p20 and two p10 subunits. The catalytic residues
in the active site are Cys
and His
. Members
of the ICE protease family have the unusual requirement for Asp in the
P1 position(21, 22) . Four amino acid side chains form
the P1 carboxylate binding pocket: Arg
,
Gln
, Arg
, and Ser
. These four
residues, as well as Cys
and His
are
conserved in ICH-2, as well as in all ICE family members reported to
date (Fig. 1B). The ICH-2 cDNA encodes a
polypeptide with 53% amino acid identity to ICE over the entire length
of the two proteins and 60% identity in the mature domains alone. ICH-2
is the most highly conserved homolog of ICE isolated to date; mature
ICE has only 27% amino acid identity with ICH-1 and only 30% with
CPP32.
Expression of ICH-2 in Human Tissues
To analyze
the expression pattern of ICH-2, a human adult multitissue
Northern blot was probed with short ICH-2- and ICE-specific
probes (264 and 347 bp, respectively) derived from the less conserved
NH-terminal regions of the coding sequences. Given the
level of sequence conservation and size similarity of the mRNAs encoded
by ICE family members, it was important to use carefully chosen, small
probes to avoid cross-hybridization between family members. As has been
observed, the ICE-specific probe hybridizes to three transcripts of
2.5,
1.7, and
0.5 kb(23) . Using an exon 6
ICE-specific probe, we do not detect the 0.5-kb message (data not
shown). The ICH-2 probe hybridizes to a single
1.7-kb mRNA (Fig. 2). The level of ICH-2 expression may be lower
than that of ICE, but the two genes show a very similar distribution
pattern; under these hybridization conditions, ICE and ICH-2 messages can be detected in all tissues probed, with the exception
of brain (although an ICE message has been detected in human
brain(24) ). Two exceptions to the relatively conserved
expression patterns are ovary and placenta; whereas ICE message is
barely expressed in these tissues, there are appreciable levels of ICH-2.
Figure 2:
mRNA expression pattern of ICH-2 in human tissues. Human adult multitissue Northern blots were
hybridized sequentially with ICH-2, ICE, and actin-specific
probes as described under ``Materials and Methods.''
Positions of size markers are indicated in kilobases. Note that caution
should be used in comparing signal intensities on one blot to another
as the actin control panel demonstrates clearly uneven
loading.
ICH-2 Induces Apoptosis
As described above,
all ICE family members reported to date have been shown to induce
apoptosis when overexpressed in various cell lines. To determine if
this was also true for ICH-2, we infected Sf9 insect cells with a
recombinant baculovirus expressing either the full-length protein or a
truncated version of ICH-2 which lacks the NH-terminal
prodomain. Analysis of insect cells infected with these baculovirus
constructs and constructs expressing full-length or truncated ICE
showed that expression of these proteins caused Sf9 cells to exhibit
the condensed morphology, cellular fragmentation, and internucleosomal
DNA cleavage characteristic of cells undergoing apoptotic cell death (Fig. 3).
Figure 3:
Apoptotic DNA fragmentation in Sf9 cells
expressing ICE or ICH-2. Genomic DNA was isolated 48 h postinfection
from Sf9 cells infected with wild type baculovirus (lanes 1)
or recombinant baculoviruses expressing the following proteins: p20 ICE (lane 2), p45 ICE (lane 3), p32 ICE (lane
4), p44 ICH-2 (lane 5), and p30 ICH-2 (lane 6).
p45 ICE and p44 ICH-2 are full-length proteins, p32 ICE and p30 ICH-2
are truncated versions lacking the NH-terminal prodomains,
and p20 ICE is the 20-kDa subunit of ICE alone (amino acids
120-297). DNA was analyzed on a 2.0% agarose gel. DNA size
markers are shown (lane M).
Expression and Characterization of N-His ICH-2
Protein
The maturational processing of ICE, perhaps performed by
ICE itself, is a result of cleavage after Asp residues 119, 297, and
316(4) . Asp and Asp
are conserved
in ICH-2 (Asp
and Asp
). The ICE Asp
site is not perfectly conserved in ICH-2, but there is an Asp at
position 104 which might define the cleavage site for removal of the
prodomain and generation of a 32-kDa form of ICH-2. Therefore, to
generate ICH-2 protein with cysteine protease activity, we subcloned
the DNA sequences encoding amino acids 105-377 of ICH-2 into an E. coli expression vector under the control of an inducible pL
promoter. By analogy with similar experiments on the ICE protein, this
region of ICH-2 was expected to be expressed in E. coli and
recovered as an active protease from the soluble fraction. As was done
for the ICE protein (see below), an NH
-terminal
polyhistidine (N-His) sequence was introduced before the putative p32
coding region of ICH-2 to facilitate recovery of the protein. N-His ICE
displays catalytic properties with peptidic substrates similar to those
of native ICE.
(
)N-His ICH-2 was expressed in E. coli and purified by nickel-chelating chromatography.
Purified N-His ICH-2 migrated on an SDS-polyacrylamide gel as two bands
with approximate molecular masses of 20 and 10 kDa (data not shown),
suggesting that the N-His ICH-2 is proteolytically processed to
subunits analogous to those of mature ICE during expression and/or
purification. Purified N-His ICE also displayed major bands at 20 and
10 kDa. In addition, gel analysis revealed partial degradation,
suggesting that a significant portion of the N-His ICE protein
preparation was inactive (data not shown). NH
-terminal
sequencing of the 10-kDa band was identical to the ICH-2 sequence
downstream of Asp
, showing that this residue may serve as
an analogous autocleavage site to Asp
of ICE. Proteolytic
activity of ICH-2 was analyzed in two ways; the N-His ICH-2 protein was
tested for the ability to cleave whole protein substrates, and its
enzymatic properties were studied in more detail using small peptide
substrates.
at two
processing sites: cleavage between Asp
and Gly
generates a 28-kDa intermediate, and cleavage between Asp
and Ala
generates the 17-kDa bioactive
product(2, 25) . To investigate the cleavage reactions
performed by the two proteases, we compared cleavage of an
[
S]methionine-labeled pro-IL-1
substrate
protein by the purified N-His ICH-2 and N-His ICE proteins. N-His ICE
processed all of the pro-IL-1
to the mature 17-kDa form by the
30-min time point, whereas incubation with N-His ICH-2 led to the
generation of only small amounts of mature IL-1
, even after 120
min (Fig. 4A). To compare the ability of the two enzymes
to generate the 28-kDa intermediate form, equal amounts of N-His ICH-2
or N-His ICE were combined with a mutant pro-IL-1
protein in which
Asp
-Ala
been changed to
Asn
-Val
. N-His ICE and N-His ICH-2 are
unable to cleave this mutated protein such that the 17-kDa mature form
of IL-1
is generated (Fig. 4B). N-His ICE rapidly
cleaves all of the mutant substrate to the 28-kDa form by 5 min; N-His
ICH-2 performs this cleavage to completion by the 60-min time point.
These data suggest that although ICE efficiently cleaves pro-IL-1
at both the Asp
and Asp
processing sites,
ICH-2 demonstrates a pronounced difference in its activity on these two
cleavage sites.
Figure 4:
Cleavage of ICE protein substrates in
vitro. A, cleavage of wild type pro-IL-1. B,
cleavage of mutant pro-IL-1
in which Asp
-Ala
has been mutated to Asn
-Val
. Equal
amounts (20 µg for pro-IL-1
cleavage, 5 µg for mutant
pro-IL-1
cleavage) of N-His ICE and N-His ICH-2 were combined with
the indicated [
S]methionine-labeled substrate
protein. Samples were removed to denaturing loading buffer at the
indicated time points and analyzed at the completion of the experiment
on a 10-20% Tris-Tricine gradient polyacrylamide gel (Integrated
Separation Systems). Positions of 31-kDa pro-IL-1
(pIL-1
), the 28-kDa intermediate (28K), the
17-kDa mature IL-1
(mIL-1
), and the 14-kDa prodomain
are indicated with arrows.
The in vitro catalytic properties of N-His
ICE and N-His ICH-2 were compared using the chromogenic peptide
substrate acetyl-Tyr-Val-Ala-Asp-p-nitroanilide(18) .
Plots of velocity versus substrate concentration (not shown)
were fit to the Michaelis-Menten equation to obtain Kvalues which were 681 ± 84
µM for N-His ICH-2 and 83 ± 10 µM for
N-His ICE. The tetrapeptide aldehyde inhibitor Ac-Tyr-Val-Asp-CHO (Ref.
4, Bachem Bioscience, Inc., King of Prussia, PA) also revealed
differences in peptide binding to the enzymes. Using the substrate
Ac-Tyr-Val-Ala-Asp-p-nitroanilide at concentrations equal to
the measured K
values for each enzyme,
Ac-Tyr-Val-Asp-CHO displayed IC
values of 38 and 748
nM for N-His ICE and N-His ICH-2, respectively. By contrast,
small molecule inhibitors had similar effects on the two enzymes. Both
were highly sensitive to the thiol reagents iodoacetamide and
iodoacetic acid, confirming their classification as cysteine proteases,
and both were resistant to the serine protease inhibitor PMSF (). E64(26) , an inhibitor of several cysteine
proteases (27) with ICE being a notable exception(25) ,
was ineffective against both. The results of these small molecule
inhibitor and peptide substrate/inhibitor studies argue that the
enzymes share catalytic similarities but differ in their recognition of
proteins in the P2-P4 positions.
(
)but apparently does
not compensate for the lack of ICE protein in ICE-deficient mice, in
the production of mature IL-1
or the response to
lipopolysaccharide-mediated sepsis (3). We demonstrate here differences
in the recognition of protein and peptide substrates and inhibitors by
ICE and ICH-2, suggesting that ICE and ICH-2 may participate in quite
different cellular functions or pathways.
Table: Inhibitor sensitivity of N-His ICH-2
/EMBL Data Bank with accession
number(s) U25804.
-converting enzyme;
hICE, human ICE; IL-1
, interleukin-1
; PCR, polymerase chain
reaction; Sf9, Spodoptera frugiperda cells; AcMNPV, Autographa californica nuclear polyhedrosis virus; N-His,
NH
-terminal polyhistidine; PMSF, phenylmethylsulfonyl
fluoride; bp, base pair(s); kb, kilobase(s); CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.