From the
The ability of cytolytic cells to cause apoptosis in target
cells is in part due to the action of the serine proteinase granzyme B.
We demonstrate that granzyme B is inhibited, with an association rate
constant of 2.9
The granzymes are a family of serine proteinases present in the
lytic granules of cytotoxic T-lymphocytes and natural killer cells
(1) . They consist of a number of enzymes, with various primary
substrate specificities, that play a role in apoptosis of susceptible
target cells
(2, 3, 4, 5) . Granzymes
must be delivered from an effector cell to the target cell, with the
help of the pore-forming protein perforin to produce their apoptotic
effect
(6) . The granzyme shown to cause the most rapid kinetics
of cell death is granzyme B
(2) , which has an unusual
preference for Asp in the primary substrate site
(7, 8) . Of the hundreds of characterized eukaryotic
proteinases, the only other enzymes that share a specificity for Asp
are interleukin-1
If apoptotic
proteinases can share primary substrate specificities it is possible
that they will be inactivated by the same inhibitors. Currently the
only known natural inhibitor of these Asp-specific proteinases is the
cowpox virus serpin CrmA that is able to rapidly inhibit ICE
(12, 13, 14) . This is consistent with the
ability of the virus to inhibit the host's inflammatory response
(13, 15) . The interaction between CrmA and ICE is
unusual because ICE is a cysteine proteinase and CrmA is from a family
of inhibitors of serine proteinases. The inhibitory site of CrmA
consists of a stretch of primary structure that mimics a good substrate
for ICE; indeed this sequence contains an Asp residue strategically
placed to bind to the primary substrate site of proteinases
(12) . Since ICE and GraB share primary substrate specificity,
we investigated whether CrmA could inhibit GraB and whether this could
constitute part of the virus' offensive strategy. GraB is a
reasonable target for inhibition by CrmA for at least two reasons.
First, it is a serine proteinase of the type to which serpins such as
CrmA are normally directed. Second, GraB is secreted into the cytosolic
compartment of virally infected target cells, where CrmA exerts its
biological role.
GraB was purified from 3
For expression of CrmA
in vitro or in Escherichia coli the full-length
crmA gene was ligated to a linker that encoded an N-terminal
Met-His
We analyzed the interaction between human GraB and CrmA using
an in vitro expression system developed for rapid screening of
serpin/proteinase interactions
(19) . Preliminary experiments
using lysates of COS cells that express mouse GraB revealed the
formation of SDS-stable complexes with CrmA and active enzyme but not
when the proenzyme was used (not shown). We performed the rest of our
studies using human GraB because it can be isolated in active pure
form, in milligram quantities, from the human YT (natural killer cell
leukemia) cell line.
Commitment to cell death is an irreversible process, and it is
reasonable to utilize proteinases since they generate irreversible
events. The proteinases identified in the endogenous cell death pathway
have a characterized or suspected preference for cleaving substrates at
Asp, a rare specificity compatible with tight regulation. Because GraB
is delivered from cells whose main function is to kill virally infected
targets (reviewed by Berke
(6) ), its inhibition by CrmA is
consistent with the biology of the cowpox virus. Variants of the virus
containing deletions in the crmA gene have an attenuated
phenotype consistent with an inability of the variants to inhibit
ICE-mediated processing of prointerleukin-1
We thank Bill Hanna and Chris Froelich for providing
advice and the YT cell line, and Ed Madison and our colleagues at Duke
for helpful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
10
M
s
, by the cowpox viral serpin cytokine
response modifier A (CrmA). Previously we have shown CrmA to be an
inhibitor of the cysteine proteinase interleukin-1
-converting
enzyme (ICE). Thus the mechanism of CrmA involves the unusual ability
to efficiently inhibit proteinases from two distinct catalytic classes,
in this case serine and cysteine proteinases. Granzyme B and ICE are
both used to combat viral infection, and we propose that cowpox virus
uses CrmA to evade the contribution of these two proteinases. Thus,
through CrmA, the virus may influence two of the pathways normally used
to kill virus-infected cells: acting on endogenous proteinases such as
ICE and on exogenous proteinases delivered by cytotoxic lymphocytes to
infected cells.
-converting enzyme (ICE),
(
)
which cleaves the Asp
-Ala
bond in prointerleukin-1
(9, 10) , and a
proteolytic activity designated prICE that cleaves poly(ADP-ribose)
polymerase between Asp
and Gly
in an in
vitro model of apoptosis
(11) . The shared primary
specificity of these proteinases is an unlikely coincidence and implies
that proteins involved in cell death pathways include key substrates
that are activated by cleavage at Asp residues.
10
YT cells by
cavitation, differential salt extraction, and ion exchange
chromatography essentially as described
(16) . Enzymatic
activity was routinely determined by using
Boc-Ala-Ala-Pro-Asp- pNA (where pNA is
p-nitroanilide) at 0.5 mM final concentration.
GraB/CrmA association kinetics were determined using the more sensitive
substrate Boc-Ala-Ala-Asp-SBzl (where SBzl is S-benzyl) with
detection of released thiobenzene by 5,5`-dithiobis(nitrobenzoic acid)
(7) . Substrate hydrolyses were recorded using a Molecular
Devices V
plate reader or a Shimadzu
spectrophotometer. All assays were performed in reaction buffer
consisting of 20 mM Hepes buffer, pH 7.4, containing 0.1
M NaCl and 0.05% Nonidet P-40.
fusion to facilitate purification. The coding
sequence started with the initiator methionine, followed by six
histidines, a serine, and then the entire coding region of CrmA. The
parent plasmid for in vitro translation was pTM1
(17) ,
and the parent for E. coli expression was based on the
isopropyl-1-thio-
-D-galactopyranoside-inducible pFLAG
(IBI). Coupled transcription/translation was performed with the
TNT
kit from Promega according to the
manufacturer's recommendations. Briefly, 0.5 µg of plasmid
DNA was incubated for 1 h at 31 °C in a total volume of 50 µl
containing the kit reagents and 20 µCi of translation grade
[
S]Met. Once translated, the reaction mix was
either used immediately or stored at -20 °C until needed.
Expression and Purification of CrmA from E. coli
E.
coli TG1 containing the CrmA construct was induced with
isopropyl-1-thio--D-galactopyranoside for 3 h and
harvested, and the cells were lysed by sonication and pelleted by
centrifugation. The supernatant containing soluble CrmA was filtered
through a 0.22-µm filter and then loaded onto a 2-ml
Ni-nitrilotriacetate column (Qiagen) and washed with 50 mM
Tris, pH 8.0, containing 0.5 M NaCl. CrmA was eluted with 50
mM Tris, 50 mM imidazole, pH 8.0, containing 0.1
M NaCl. This material was diluted with 9 volumes of 20
mM Hepes, pH 7.4, containing 2 mM dithiothreitol and
applied to a 2-ml column of DEAE-Sepharose. This column was developed
with a linear gradient of 0-1 M NaCl in 20 mM
Hepes buffer, pH 7.4, and CrmA was eluted at approximately 0.4
M NaCl to produce a yield of 4 mg of protein from 6 liters of
culture fluid. The material was greater than 95% pure as estimated by
Coomassie Blue staining and was stored at 4 °C until use. In all
experiments using this material the CrmA was treated with 2 mM
dithiothreitol for 5 min, followed by 4 mM NaAsO
to block excess dithiothreitol. This procedure resulted in CrmA
with the highest inhibitory activity.
Polyacrylamide Gel Electrophoresis (PAGE)
CrmA
protein samples were analyzed by non-denaturing PAGE or reduced
SDS-PAGE in gels of 5-15% linear acrylamide gradients
(18) . For radioactive samples, the gels were dried and then
subjected to autoradiography using a phosphorescent image scanner
(PhosphorImager 410A and densitometer, Molecular Dynamics, Sunnyvale,
CA); otherwise, protein bands were visualized by R250 Coomassie
staining.
(
)
The majority of in
vitro translated CrmA formed a complex with GraB that did not
migrate in native gel electrophoresis due to the highly basic
composition of GraB (Fig. 1 A, top). This
complex is resolved on SDS-PAGE, a phenomenon typical of
serpin/proteinase interactions (Fig. 1 A,
bottom).
Figure 1:
Specificity of the
interaction of GraB with CrmA. PanelA, complex
formation between CrmA and GraB. [S]Met-labeled
CrmA was incubated with dilutions of unlabeled purified human GraB for
30 min at 37 °C in a total volume of 20 µl. The reactions were
divided and then run on native PAGE ( top) and reduced
SDS-PAGE ( bottom), and the products were quantitated by
PhosphorImaging. Because the complex does not migrate into a native
gel, the formation of the CrmA
GraB complex can be seen as the
disappearance of CrmA ( arrowhead). However, SDS-PAGE allows
visualization of the CrmA
GraB complex ( arrow) since all
components migrate in the gel when denatured. The intermediate band
seen in some lanes is probably partially degraded complex.
PanelB, activity of GraB. Purified human GraB (85
pmol) was allowed to react with a 3-fold molar excess of recombinant
CrmA in a total volume of 50 µl and then run on reducing SDS-PAGE,
followed by staining with Coomassie Blue. Lane1,
GraB alone; lane2, the same amount of GraB reacted
with CrmA; lane3, CrmA alone. Virtually all of the
GraB shifted to form an SDS-stable complex with CrmA, indicating that
the enzyme is fully active. The lowerband in the
CrmA sample probably represents a small amount of cleaved inhibitor
present in the preparation.
PhosphorImager quantitation of the radioactive
bands indicated that about 95% of CrmA was able to form a complex with
GraB. Similarly, virtually all of the purified GraB formed a complex
with CrmA (Fig. 1 B). To characterize this apparent
inhibition, we titrated GraB activity with CrmA produced in E. coli (Fig. 2) and observed linear concentration-dependent
inhibition of GraB, indicative of a tight binding interaction. Under
the conditions used here, the binding to CrmA was too tight to measure
equilibrium, so we explored the reaction in more detail by observing
the rate of inhibition (Fig. 2). This was linear over at least
three half-lives of the reaction, indicating standard bimolecular
kinetics. Thus we demonstrate for the first time that CrmA can
efficiently inhibit a serine proteinase. Previously we showed that CrmA
is an efficient inhibitor of ICE, a cysteine proteinase
(14) .
Our current data now firmly establish that CrmA, since it is able to
inhibit a serine proteinase and a cysteine proteinase, is a cross-class
inhibitor. This unusual property would be explained by ICE having a
substrate binding geometry that more closely resembles serine
proteinases than other cysteine proteinases.
Figure 2:
Kinetics of the interaction of GraB with
CrmA. PanelA, GraB titrated by CrmA. Purified GraB
was incubated with the indicated concentrations of recombinant CrmA at
37 °C in reaction buffer. After 15 min
Boc-Ala-Ala-Pro-Asp- p-nitroanilide was added to determine the
residual GraB activity. The active enzyme concentration was calculated
based on the concentration of CrmA determined as described before (13).
PanelB, determination of the rate of inhibition of
GraB by CrmA. CrmA was incubated with equimolar GraB (1.7
10
M) at 37 °C in reaction buffer.
Portions were removed at timed intervals and assayed for residual GraB
activity via a coupled assay with Boc-Ala-Ala-Asp-SBzl (where SBzl is
S-benzyl) and 5,5`-dithiobis(nitrobenzoic acid). The plot of
reciprocal residual enzyme concentration against time was linear,
indicating a bimolecular reaction, with a slope of 2.9
10
M
s
.
The rate of inhibition
of GraB by CrmA, 2.9 10
M
s
, is certainly fast enough to be of
physiologic significance. For example it exceeds the rate of inhibition
of plasma kallikrein by the serpin C1-inhibitor
(20) . However,
the rate is almost 2 orders of magnitude slower than the inhibition of
ICE by CrmA, which at 1.7
10
M
s
is among the
fastest of serpin/target proteinase interactions. A comparison of the
rates of inhibition predicts that ICE would compete for binding to CrmA
about 60 times better than would GraB, and we tested this by incubating
CrmA with a mixture of the two proteinases at different molar ratios
(Fig. 3). The ICE
CrmA complex is not stable to SDS but is
observed in native gels (Fig. 3 A). Conversely the
GraB
CrmA complex is stable to SDS, so the resulting competition
can be distinguished by observing complexes in native and SDS gels. The
equivalence point is in lane4, representing a
GraB/ICE ratio of 100:1. This is in agreement with the rate
determinations and demonstrates that CrmA, though it is an adequate
GraB inhibitor, will preferentially inhibit ICE.
Figure 3:
Competition between GraB and ICE for
binding to CrmA. [S]Met-labeled CrmA was reacted
with buffer ( lane1) or serial 3-fold dilutions of
ICE ( lanes2-7) in a total volume of 20 µl
( A). About 35% of the CrmA formed a complex with ICE, and the
rest was turned over as described previously for this reaction (14).
CrmA was allowed to react with GraB (42.5 pmol) in the presence of the
serial dilutions of ICE in a total volume of 30 µl ( B).
The reactions were divided and then run on native PAGE and reduced
SDS-PAGE. The reactions were carried out in 0.1 M NaCl, 10%
sucrose, 0.1% Chaps, and 50 mM Hepes buffer, pH 7.4, at 37
°C for 30 min. After PAGE the gels were dried and then analyzed by
PhosphorImaging.
Our findings have
implications for the mechanism of proteinase inhibition by serpins and
the biological role of CrmA. Previously we had shown that CrmA is an
inhibitor of the cysteine proteinase ICE, and now we show that it is
also an inhibitor of the serine proteinase GraB. Since both proteinases
compete for binding to CrmA they most likely react with the same
inhibitory site. Thus this serpin can inactivate structurally unrelated
proteinases, provided they share the same primary substrate
specificities. How CrmA accomplishes this is still a puzzle, but this
adaptability has important implications for other members of the ICE
family, including Ced-3
(21) , CPP32
(22) , and Ich-1
(23) (the human ortholog of Nedd-2
(24) ), that have been
implicated as positive regulators of apoptosis. The substrate
specificity of these putative proteinases has yet to be reported,
though they contain the residues that impart Asp specificity to ICE,
and it is predicted that they will cleave protein substrates required
for progression of the death pathway inherent to a number of cell
types. Thus they are candidates for the CrmA-inhibitable mediator(s) of
programmed cell death implicated in a number of recent studies based on
cell transfection strategies
(21, 25, 26) .
. Our data on GraB
inhibition suggest a second role for CrmA. Since virally infected cells
can be cleared from the body by cytolytic cells using their
perforin/granzyme system, an additional function of CrmA may be to
prevent the activation of apoptosis via the GraB pathway. Thus we raise
the possibility that, through the CrmA serpin, the virus has acquired
the ability to manipulate the cell death program by inhibiting
endogenous and exogenous mediators.
-converting enzyme;
CrmA, cytokine response modifier A; GraB, granzyme B; PAGE,
polyacrylamide gel electrophoresis; Boc, t-butoxycarbonyl;
Chaps,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.