From the
The interleukin-1
Interleukin-1
Strains and Plasmid Construction The complete precursor ICE (p45) cDNA was cloned, via polymerase chain
reaction amplification, from a commercially available human peripheral
blood monocyte cDNA library (Clonetech 1050a), into vector
pGI-
From 100 liters of fermentation broth, a 155-g wet cell
pellet was obtained from which 22 g of inclusion bodies were isolated.
At this stage, the purity of the p45 was no more than 25% (Fig.
2 a). Following solubilization and reversed-phase HPLC, the
purity was increased to more than 80%, with p45 being observed
essentially as a single band on SDS-PAGE (Fig. 2 a), which
remained present following dilution to 2
M urea and the first
dialysis. But during the second dialysis, this band gradually
disappeared, being replaced by a large number of bands ranging in
molecular mass from 36 to 8 kDa that had not been present in the
starting material. Two of the most intense bands corresponded in
molecular mass to the p10 and p20 subunits of ICE
(Fig. 2 a). The gel was blotted onto polyvinylidine
difluoride, and the most intense bands were sequenced. A total of seven
bands with molecular masses of 45, 36, 27, 25, 23, 14, and 12 kDa,
respectively, were cut and sequenced from the polyvinylidine difluoride
membrane. The results (Figs. 3, a and b) show that
all of the bands contained either the p45 N-terminal sequence or
sequences generated following cleavage of an Asp- X bond.
The bands at 25, 23, and 12 kDa were identified as p24, p20, and p10,
respectively. The bands at 45 and 36 kDa contained only the p45
N-terminal sequence; the band at 36 kDa must, therefore, have been
generated by cleavage at the C-terminal end of the protein. The band at
14 kDa consisted primarily (66%) of an elongated form of p10 containing
the Ser
We have demonstrated that, following refolding by dilution
and subsequent dialysis, purified recombinant p45 ICE is converted to
p10/p20 ICE. Conversion occurs in a time-dependent manner through a
series of intermediates. Identification of these through sequence
analysis has given us an insight into the mechanism of conversion. The
process begins slowly during the second dialysis step as the chaotroph
concentration is reduced and three prominent products are seen. These
are a 36-kDa C-terminally truncated p45, p10, and p10 carrying the
Ser
The reaction that we have observed has been carried out
in vitro using p45 expressed as insoluble inclusion bodies in
E. coli. These inclusion bodies were purified under strongly
denaturing conditions prior to refolding and would therefore be
unlikely to have contained active proteases of bacterial origin. In a
control experiment (not shown) an inclusion body fraction from the same
host cell line, but expressing an unrelated protein was enriched using
HPLC and a fraction corresponding to the position of elution of p45ICE
collected and refolded using identical conditions to those used for p45
ICE. No protease activity was detected using the IL-1
Following dilution under reducing conditions,
dialysis carried out in the absence of GSH, DTT, or
When p45 was observed to be
expressed in an intact form in a rabbit reticulocyte lysate system
(9) , this stability may have been due to the relatively low
thiol content of the translation mixture. How ICE autocatalysis is
controlled in vivo remains to be seen. However, the fact that
an N-terminally truncated form (minus amino acids 1-119) of the
mouse enzyme expressed in E. coli was produced as a p10/p20
heterodimer indicates a role for amino acids 1-119 in the
regulation of autocatalysis. We have shown that removal of amino acids
1-119 is not necessary for autocatalysis to occur and that the
regulatory mechanism can, in-vitro, be bypassed by high
concentrations of low molecular weight thiols. The strong effects
exerted by thiols suggest that the three closely located cysteines at
positions Cys
We have shown that the
interleukin-1
-converting enzyme is a heterodimeric
cysteine protease that is produced as a 45-kDa precursor. The
full-length precursor form of the enzyme was expressed in
Escherichia coli as insoluble inclusion bodies. Following
solubilization and refolding of the 45-kDa protein, autoproteolytic
conversion to a heterodimeric form containing 10- and 20-kDa subunits
was observed. This enzyme had catalytic activity against both natural
(interleukin-1
precursor) and synthetic peptide substrates. The
inclusion of a specific inhibitor (SDZ 223-941) of the converting
enzyme in the refolding mixture prevented proteolytic processing to the
10-/20-kDa form. Similarly, refolding under nonreducing conditions also
prevented processing. Time course experiments showed that the 10-kDa
subunit was released from the 45-kDa precursor before the 20-kDa
subunit, implying that the N-terminal portion of the precursor is
released last and may play a regulatory role.
(IL-1
)
(
)
is a
potent mediator of inflammation. IL-1
is produced in monocytes and
monocytic cell lines as an inactive 31-kDa precursor
(1, 2) . The active 17.5-kDa polypeptide is generated by
specific proteolytic cleavage of the precursor molecule at
Asp
-Ala
(3, 4) . It
has been demonstrated that in human monocytes, proteolytic processing
of the IL-1
precursor is carried out by a specific protease known
as the IL-1
-converting enzyme (ICE)
(5, 6, 7, 8) . ICE has recently been
purified, characterized, and cloned
(8, 9, 10) and shown to be a heterodimeric cysteine protease
(5, 9, 11) , comprising 10- (p10) and 20-kDa
(p20) subunits in a 1:1 stoichiometric ratio. Structural data
(12, 13) show that the active form of the enzyme is a
tetramer formed from two of the p10/p20 heterodimers. The heterodimeric
form is generated from a 45-kDa precursor (p45) by the proteolytic
cleavage of four peptide bonds (Asp
-Ser
,
Asp
-Asn
,
Asp
-Ser
, and
Asp
-Ala
; see Fig. 1)
(8, 9) . ICE has an unusual specificity, requiring
aspartic acid at the position N-terminal to the scissile peptide bond
(P1)
(9, 10, 14, 15) . This P1
specificity is shared with only two other enzymes from mammalian
sources, granzyme B
(16) , a serine protease from cytolytic
lymphocytes, and prICE
(17) , an enzyme that cleaves
poly(ADP-ribose) polymerase. This very rare specificity suggests that a
mechanism of autocatalytic processing is responsible for the generation
of mature p10/p20 ICE. This theory is supported by several pieces of
experimental evidence. The addition of purified ICE to the intact p45
precursor results in the generation of p10/p20 ICE
(9) ; p45
expressed in the cytosolic fraction of SF9 cells using the baculovirus
system is converted to the p10/p20 heterodimer form
(18) , and a
truncated form of the mouse ICE enzyme (minus amino acids 1-119)
is expressed in Escherichia coli as a p10/p20 heterodimer
(10) . Surprisingly, however, the p45 precursor, when expressed
in rabbit reticulocyte lysate, remains stable
(9) .
Figure 1:
Primary structure of the human
IL-1-converting enzyme precursor. Full-length p45 ICE may be
cleaved at the four Asp- X cleavage sites indicated to
produce mature heterodimeric p10/p20 ICE ( stippled regions). Cleavage at Asp
-Ser
and Asp
-Ser
results in the
formation of the polypeptides designated p24 and
p14.
Here we
show conclusively that following expression, purification, and
refolding, the p45 precursor autocatalytically converts to the mature
p10/p20 form of ICE.
-PBR#13, under the control of the
PL promoter and
transfected into the E. coli strain SG936pCI857. The p45 cDNA
clone was sequenced, and the DNA sequence was shown to agree with that
reported by Thornberry et al. (9) . Fermentation of the Expression Strain The SG936pCI857 expression strain was grown in 1 liter of
L-broth (NaCl, 10 g/liter; Difco Bacto-tryptone, 10 g/liter;
BBL Bacto yeast extract, 5 g/liter), supplemented with ampicillin (0.1
g/liter), and kanamycin (0.04 g/liter) for 6 h at 30 °C. This
preculture was used to inoculate 19 liters of fermentation medium
(
L-broth with ampicillin, 0.1 g/liter) in a 30-liter steel
fermenter. The bacteria were grown at 30 °C at an agitation speed
of 200 rpm and an aeration rate of 1 (v/v/min). The expression of p45
ICE was induced by a temperature shift to 42 °C when an
A
value of 0.5-0.7 had been reached. The
cells were harvested 4 h after induction; at which time, no further
increase in A
above a value of 3-4 was
observed. The culture was harvested by centrifugation, and the cell
paste was frozen until required. Inclusion Body Isolation and Purification Frozen E. coli wet cell pellets were suspended to 12.5% (w/v)
in cell lysis buffer (50 m
M Tris, pH 8.0; containing 2
m
M DTT, 5 m
M benzamidine-HCl, and 2 m
M EDTA)
and mixed by stirring for 1 h on ice. The cell suspension was lysed by
passage through a Manton-Gaulin homogenizer (2 passes at 1200 bar) and
then centrifuged for 30 min at 16,000
g. The resultant
pellets were resuspended in lysis buffer and recentrifuged a further 3
times. Semipurified inclusion bodies were solubilized in 6
M
guanidine-HCl, 25 m
M DTT and purified by sequential
preparative reversed phase HPLC on two Orpegen HD gel RP-7 s C8 (22
250 mm) columns using a water/acetonitrile buffer system. p45
containing fractions from the second Orpegen column were pooled and,
following removal of organic solvent, lyophilized and stored at
-20 °C. Refolding of p45 Lyophilized inclusion bodies were solubilized (1 mg/ml) in 50
m
M Tris, pH 8.0 (containing 8
M urea, 50 m
M
DTT), for 1 h at room temperature. Following dilution with 3 volumes of
50 m
M Tris, pH 8.0, 10 m
M DTT, the refolding mixture
was stirred overnight at room temperature and then dialyzed overnight
at 4 °C against 2
80 volumes of 50 m
M Tris, pH
8.0, containing 10 m
M GSH. Refolding and p45 purification were
monitored by analytical reversed phase HPLC using a 4.6
150-mm
Polymer Laboratories PLRP-S 1000-Å column. Refolding in the Presence of a Specific ICE Inhibitor Refolding in the presence of SDZ 223-941, a P-site substrate based
irreversible inhibitor, was carried out as described above except that
at the dilution and dialysis steps inhibitor (SDZ 223-941 10
mg/ml stock in Me
SO) was added to a 1.5 molar excess.
Samples were removed at each step for SDS-PAGE and HPLC analyses. Gel Filtration Chromatography 4 ml of concentrated ICE was loaded onto a 1.6
60-cm column of
Superdex 75 equilibrated in phosphate-buffered saline containing 10
m
M DTT. 1-ml fractions were collected and analyzed on
SDS-PAGE, and those found to contain p10/p20 ICE were pooled and
tested. IL-1
Convertase Activity Determinations
Activity Determinations Using Recombinant hu-IL-1
ICE activity was detected by monitoring
cleavage of recombinant IL-1
Precursor as Substrate
precursor to mature IL-1
using
analytic HPLC. ICE samples were added (1:100 enzyme/substrate ratio) to
1 ml of recombinant hu-IL-1
precursor solution (330 µg/ml in
phosphate-buffered saline, 10% glycerol) and incubated for 2 h at 37
°C. The incubation mixtures were then diluted 3-fold and analyzed
on reversed phase HPLC (80 µl injection volume). Untreated
precursor and recombinant hu-IL-1
were used as standards.
SDS-PAGE
SDS-PAGE was carried out under reducing
conditions on 4-20% Novex gradient gels run according to the
manufacturer's instructions.
N-terminal Sequence Analysis
N-terminal amino acid
sequence determination by Edman degradation was performed on an Applied
Biosystems 470A protein sequencer fitted with an HPLC on-line system
120A. Protein mixtures were run on 4-20% gradient gels (Novex)
and electrophoretically transferred to polyvinylidine difluoride
membranes
(19) . Following identification of the protein bands,
the blots were completely destained with 100% methanol, and the protein
bands were cut out (1 10-mm pieces). These were then loaded
into a continuous flow reactor
(20) and microsequenced.
-Asp
linker (see Figs. 1 and
3 b). The band at 27 kDa contained 5 sequences; one identified
as the p45 N-terminal (11%) and the remainder as fragments generated by
cleavage after Asp
(49%), Asp
(15%),
Asp
(13%), and Asp
(12%). When samples of
the refolding mixture were removed at different times during the
refolding and analyzed using SDS-PAGE, it was observed
(Fig. 2 b) that the 36 and 14 kDa bands were generated
first, followed by the bands at 27, 25, 23, and 12 kDa.
Figure 2:
Recombinant human p45 ICE
autocatalytically converts to p10/p20. Analysis of the autocatalytic
conversion using reducing SDS-PAGE (Novex 4-20%) is shown.
a, purification and autocatalytic cleavage. Lane 1, low molecular mass marker (Pharmacia Biotech Inc.);
lane 2, p45 ICE inclusion body preparation; lane 3, p45 ICE purified from inclusion bodies by preparative
reversed phase HPLC; lane 4, purified p45 ICE diluted
down to 2
M urea prior to dialysis; lane 5,
p45 ICE solution following dialysis at 4 °C against 2 80
volumes of dialysis buffer (50 m
M Tris, pH 8.0; containing 10
m
M GSH); lane 6, p10/p20 ICE purified on
Superdex 75. b, the time dependence of autocatalytic
conversion. Following dialysis against 2
100 volumes of
dialysis buffer (in the absence of reducing agents), the retentate was
concentrated 10-fold (2 m
M oxidized glutathione was added to
prevent autocatalysis during concentration); DTT was then added to 20
m
M, and samples removed for SDS-PAGE at appropriate time
intervals. Lane 1, molecular mass markers; lane 2, sample taken before dialysis; lane 3, sample taken after dialysis; lane 4,
concentrated sample on addition of DTT; lane 5,
sample 1 min after DTT addition; lane 6, sample 5 min
after DTT addition; lane 7, sample 10 min after DTT
addition; lane 8, sample 15 min after DTT addition;
lane 9, sample 30 min after DTT addition; lane 10, sample 45 min after DTT addition. c, the
influence of different reducing agents on the rate of autocatalysis and
the effect of a specific ICE inhibitor on autocatalysis. Lane 1, molecular mass markers; lane 2, p45
ICE solution (200 µg/ml p45 in 2
M urea, 50 m
M
Tris, 10 m
M DTT, pH 8.0) prior to dialysis; lane 3, p45 ICE solution dialyzed against 50 m
M Tris,
10 m
M GSH, pH 8.0; lane 4, p45 ICE solution
dialyzed against 50 m
M Tris, 10 m
M DTT, pH 8.0;
lane 5, p45 ICE solution dialyzed against 50
m
M Tris, 10 m
M
-mercaptoethanol, pH 8.0;
lane 6, p45 ICE solution dialyzed against 50
m
M Tris, pH 8.0, containing no reducing agents; lane 7, p45 ICE solution (containing a 1.5 molar excess of the
specific ICE inhibitor SDZ 223-941) following dialysis against 50
m
M Tris, pH 8.0 (containing 10 m
M GSH and a 1.5 molar
excess of 223-941); lane 8, p45 ICE solution
dialyzed against 50 m
M Tris, pH 8.0 (no reducing agents) and
then made 10 m
M with respect to GSH and incubated for 2 h at
room temperature.
When
analyzed on SDS-PAGE, refolding mixtures containing the specific ICE
inhibitor SDZ 223-941 showed a greatly reduced generation of
lower molecular weight species, in contrast to what was observed in
reactions containing only GSH, DTT, or -mercaptoethanol
(Fig. 2 c). Similarly such refolding mixtures were shown
by analytical HPLC to be inactive using recombinant IL-1
precursor
as substrate (Fig. 4 d). When the dialysis step of refolding
was carried out under nonreducing conditions by omission of the thiol
reagents, p45 remained similarly undegraded. Subsequent addition of GSH
to 10 m
M resulted in rapid breakdown of p45 (Fig.
2 c). When GSH in the dialysis buffer was replaced by either
-mercaptoethanol or DTT at the same concentration, a similar
generation of lower molecular weight fragments was observed (Fig.
2 c). However, dialysis with DTT led to a further degradation
of p10 not seen with either GSH or
-mercaptoethanol. Incubation of
recombinant hu-IL-1
precursor with p45 solution after dialysis was
shown by HPLC (Fig. 4 c) to result in an almost complete
breakdown of IL-1
precursor yielding mature IL-1
and three
precursor fragments. Determination of the ICE activity of this
preparation using a synthetic peptide substrate revealed a specific
activity of 0.369 µmol/mg/min (not shown). When a concentrated
solution of p45 dialyzed under reducing conditions was run on Superdex
75, a peak was eluted with an apparent molecular mass of 34,500 daltons
that was shown by SDS-PAGE (Fig. 2 a) to contain
virtually pure p10/p20. When tested against recombinant IL-1
precursor, this material was shown to possess ICE activity
(Fig. 4 e).
Figure 4:
Specific ICE activity of autocatalytically
processed p45. Analytical reversed phase HPLC of different p45 ICE
fractions incubated at 37 °C for 2 h with a 100-fold excess of
recombinant hu-IL-1 precursor (500 µg/ml in phosphate-buffered
saline, 10% (v/v) glycerol) and then diluted 3-fold with HPLC buffer A.
The positions of IL-1
, the IL-1
precursor, and fragments
derived following cleavage of the IL-1
precursor are indicated.
a, control profile, r-hu-IL-1
precursor; b,
control profile, r-hu-IL-1
; c, specific ICE activity of
p45 ICE dialyzed against 50 m
M Tris, 10 m
M GSH, pH
8.0. d, specific ICE activity of p45 ICE diluted and dialyzed
in the presence of a 1.5 molar excess of the ICE inhibitor SDZ
223-941. e, specific ICE activity of autocatalytically
derived p10/p20 ICE purified by chromatography on Superdex
75.
-Asp
linker. At this stage, little
or no p20 or p24 are observed. This suggests that the first step in the
conversion is the removal of p10 plus linker by cleavage at Asp
followed rapidly or simultaneously by the removal of the linker
by cleavage at Asp
. The p36 band, which contains only the
p45 N-terminal sequence, must therefore represent p45 minus
Ser
-His
and is itself further
processed down to p20 (presumably via p24, as the intensities of the
p24 and p20 bands do not increase significantly until p14 and p10 have
already been formed and p36 starts to decrease in intensity; Fig.
2 b). Therefore, the p10/p20 form is clearly formed via
processing through several intermediates, each itself being formed by
cleavage at Asp- X (Fig. 5). Conversion to p10/p20 was
abolished by the inclusion of SDZ 223-941, a P-site
substrate-based irreversible inhibitor of ICE. The end product of the
conversion was an ICE preparation of far greater stability and higher
specific activity than ICE produced by the co-refolding of separately
expressed p10/p20 subunits.
(
)
Gel-filtration
chromatography resulted in preparations containing virtually pure
active ICE with an apparent molecular mass of 34,500 Da, similar to the
previously reported figure of 29,000 Da
(13) . Lower molecular
mass protein products caused by further autodegradation upon
concentration of ICE
(12, 13) were effectively removed.
Figure 5:
The
autocatalytic processing of p45 to p10/p20 ICE occurs in an ordered
manner through several intermediates. Sequential positions of cleavage
are marked with an arrow. The stippled and shaded regions represent the polypeptides removed by autocatalytic
cleavage. a, intact p45 ICE; b, cleavage at
Asp-Ser
, resulting in the formation
of p36 and p14; c, cleavage at
Asp
-Ala
in p14, releasing p10;
d, cleavage at Asp
-Ser
in
p36, releasing p24; e, cleavage at
Asp
-Asn
in p24, releasing
p20.
Until now, the evidence for the autocatalytic conversion of p45 ICE
to p10/p20 ICE has been strong but circumstantial
(8, 9, 10, 11) . The expression of p45
in the cytosolic fraction of SF9 cells and the subsequent detection of
a number of lower molecular weight components including p10/p20 ICE
(18) , could conceivably have been due to the proteolytic
``activation'' of p45 by SF9 proteases. Earlier work
(9) had shown that p45 produced in a rabbit reticulocyte lysate
system remained in the precursor form until mature ICE had been added,
leading the authors to believe that another protease may have been
required to activate p45. The expression of a truncated form (minus
amino acids 1-119) of murine p45 in E. coli resulted in
the production of p10/p20
(10) , suggesting that the N-terminal
119 amino acids of p45 may play a regulatory role in autocatalytic
conversion.
precursor
and synthetic peptides as substrates (data not shown). The cleavage
products that we observed were those produced by cleavage at the known
cleavage sites in p45
(8, 9) . Only the faint band at 27
kDa contained sequences that were produced by cleavage at other
Asp- X sites. It has been reported that although the
protein prICE recognizes the same tetrapeptide cleavage site as ICE,
prICE does not cleave the IL-1
precursor, nor does ICE have any
activity against the prICE substrate, poly(ADP-ribose) polymerase
(17) . It is extremely unlikely that a contaminating protease
with Asp- X specificity could have been responsible for
the specific cleavage observed and that this cleavage was therefore
autocatalytic.
-mercaptoethanol prevented autocatalytic processing. The
subsequent addition of GSH resulted in rapid autocatalysis, showing
clearly that reducing agents play a key role in triggering the
autocatalytic conversion of p45 to ICE.
, Cys
, and Cys
could play a role in the regulation of enzyme activity. On the
other hand, the dependence of ICE autocatalysis on low molecular weight
thiols might merely be a reflection of the fact that mature p10/p20 ICE
requires DTT concentrations in the order of 10 m
M for
reactivation
(21) .
-converting enzyme is produced by an autocatalytic
conversion of the 45-kDa precursor in vitro. The mechanism
controlling autocatalytic conversion remains unclear, but our results
show that low molecular weight thiols such as glutathione play a key
role in triggering autocatalytic conversion in vitro.
, interleukin-1
; hu-IL-1
,
human IL-1
; ICE, IL-1
-converting enzyme; DTT,
1,4-dithio-
DL-threitol; HPLC, high performance liquid
chromatography; PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.