(Received for publication, January 31, 1996; and in revised form, March 12, 1996)
From the
Granzyme B plays an essential role in cytotoxic T lymphocyte
(CTL)-mediated cell killing. Recent studies suggest that granzyme B may
exert its effect by cleaving and activating CPP32, a member of the
interleukin-1-converting enzyme/Ced-3 family of cysteine
proteases. We have examined the processing and activation of CMH-1, a
close homologue of CPP32, by granzyme B in vitro. We have
found that granzyme B specifically cleaves CMH-1 at
Asp
-Ser
between the p20 and p12 and
activates the cysteine protease. Cleavage between p20 and the
prosequence of CMH-1 at Asp
-Ala
is
autocatalytic and is not required for CMH-1 activity in vitro.
The cleavage and activation of CMH-1 by granzyme B in vitro suggest that, in addition to CPP32, CMH-1 may also play a role in
CTL-mediated cell killing.
Cytotoxic T lymphocytes (CTLs) ()contribute to host
cell-mediated immunity against viral pathogens, parasites, and
neoplastic transformation as well as to host pathology associated with
transplant rejection and autoimmune diseases (for review see (1) ). CTLs destroy target cells in part by secreting perforin
along with a family of serine proteases, the granzymes, into the
intercellular space between the effector and target cell. Perforin, a
pore-forming protein, facilitates the entry of the granzymes into
target cells where they trigger apoptosis by an unknown
mechanism(2) . One of the granzymes, granzyme B, is a serine
protease with a substrate specificity for aspartate at P1 and is
essential for the rapid induction of DNA fragmentation and apoptosis in
target cells(3) .
Recently, Darmon et al.(4) reported that granzyme B can cleave and activate
CPP32, a member of the interleukin-1-converting enzyme (ICE)/Ced-3
family of cysteine proteases that also has a substrate specificity for
Asp-X. CPP32 has been isolated based on its ability to cleave
poly(ADP-ribose) polymerase (PARP)(5) , a DNA repair enzyme
that is rapidly proteolyzed in apoptotic cells(6) . It has
become more apparent that the ICE/Ced-3 family of proteases plays a
critical role in mediating apoptosis (see (7) for review).
Genetic studies in the nematode Caenorhabditis elegans indicate that the ced-3 gene is indispensable for cell
death during development(8) . Inhibitors of ICE-like proteases
can prevent mammalian cell death induced by a variety of
factors(5, 9, 10) . Furthermore, thymocytes
derived from ICE-deficient mice are resistant to Fas-induced cell
death, suggesting a physiological role for ICE in Fas-mediated
apoptosis(11) . Thus the regulation of these proteases may be
critical in executing apoptotic cell death.
Each of the ICE/Ced-3 family of proteases is synthesized as an inactive precursor polypeptide requiring proteolytic cleavage at specific aspartate residues to produce two subunits of approximately 20 kDa (p20) and 10 kDa (p10), which together form the active protease (see (12) ). The precursor proteins contain N-terminal prosequences of various lengths followed by the p20 and p10 subunits. In some members of the protease family, such as ICE, a short linker sequence exists between the p20 and p10 subunits. Processing and activation of these proteases in vivo remain unclear. Some precursors seem to undergo autocatalytic cleavage; others may require distinct aspartate-specific proteases for activation. Therefore, identification of CPP32 as a substrate for granzyme B suggests CTL-mediated cell death may be initiated by a protease cascade involving the ICE/Ced-3 family of proteases.
Recently, a new member of the ICE/Ced-3 family of proteases, CMH-1 (also named MCH-3 and ICE-LAP3), has been identified(13, 14, 15) . It has the highest homology to CPP32 and is able to cleave PARP and induce apoptosis in COS cells. In this paper, we examined the processing and activation of CMH-1 by granzyme B in vitro. We report that granzyme B cleaves CMH-1 specifically between p20 and p12 and activates the CMH-1 protease. The removal of the prosequence is an autocatalytic event and is not required for CMH-1 activity in vitro. The cleavage and activation of CMH-1 by granzyme B in vitro suggests that, in addition to CPP32, CMH-1 may also play a role in CTL-mediated cell killing.
Figure 1:
CMH-1
is cleaved more efficiently than CPP32 by granzyme B in vitro. A, in vitro translated S-labeled CMH-1 (lanes 1-3) or CPP32 (lanes 4-6) was
incubated for 30 min with buffer (lanes 1 and 4) or
with 4 µl of crude lysates (35 µg of protein) from mock
transfected (lanes 2 and 5) or granzyme B-transfected
COS cells (lanes 3 and 6) as described under
``Materials and Methods.'' Cleavage products were analyzed by
SDS-PAGE (16%) and fluorography. Numbers on the left represent molecular mass in kilodaltons. Positions of the
uncleaved CMH-1 (35 kDa) and CPP32 (33 kDa) are indicated on the right along with their respective cleavage products (CMH-1, 23
and 12 kDa; and CPP32, 21 and 12 kDa). B, 2 µg of purified
(His)
-tagged CMH-1-C186S (lanes 1-6) and
CPP32-C163S (lanes 7 and 8) were incubated with the
indicated amounts of the purified granzyme B (GranB) for 1 h
at 37 °C and analyzed by SDS-PAGE and Coomassie Blue staining. Lane M, molecular mass markers in kilodaltons. Positions of
uncleaved CMH-1 and CPP32 were indicated along with their cleavage
products (open arrowheads).
To compare the ability of granzyme B to cleave CPP32, we
incubated CPP32 produced by in vitro translation with the COS
cell lysates and found that the 33-kDa CPP32 was cleaved into 21- and
12-kDa peptides, as observed by Darmon et al.(4) .
Interestingly, we found that CMH-1 appeared to be cleaved more
efficiently than CPP32 by granzyme B (Fig. 1A). To
confirm this result, we purified granzyme B from granzyme B-expressing
COS cells and then incubated equal amounts (2 µg) of purified
(His)-tagged CPP32-C163S or CMH-1-C186S proteins (see below
and ``Materials and Methods'') with various amounts of the
purified enzyme. We found that CPP32 cleavage required a
25-50-fold higher concentration of granzyme B than CMH-1 cleavage (Fig. 1B). Therefore, under our experimental
conditions, CMH-1 is a superior substrate for granzyme B than CPP32.
Figure 2:
Purification of (His)-tagged
CMH-1 from E. coli. (His)
-CMH-1 was expressed and
purified as described under ``Materials and Methods,''
analyzed by SDS-PAGE on a 16% gel, and visualized by Coomassie Blue
staining. Lane 1, molecular mass marker; lane 2,
crude cell lysates (140 µg); lane 3, nickel-agarose column
flow-through (140 µg); lane 4, nickel-agarose eluate (7
µg); and lane 5, DEAE-Sepharose eluate (5 µg). Lane 6, DEAE-Sepharose eluates rechromatographed on
nickel-agarose column (4 µg).
Purified CMH-1 (2 nM) had no detectable protease activity against PARP produced by in vitro translation (Fig. 3, B and C), a known substrate for mature CMH-1(13, 14) . To determine if cleavage of CMH-1 by granzyme B would lead to its activation, CMH-1 was treated with purified granzyme B (Fig. 3B) and then incubated with the PARP substrate. We found that PARP cleavage was readily detected at nanomolar CMH-1 concentrations and was inhibited by Ac-DEVD-CHO (Fig. 3C). Since granzyme B itself had no detectable activity against PARP (Fig. 3C), these results indicate that cleavage of CMH-1 by granzyme B leads to the activation of the CMH-1 protease.
Figure 3:
Processing and activation of CMH-1 by
granzyme B. A, schematic drawing of (His)-CMH-1
with processing sites (D23-A24 and D198-S199) and
resulting fragments indicated. B, purified CMH-1 (2 µg)
was incubated for 30 min at 37 °C with 0.3 unit of purified
granzyme B or buffer in the presence or absence of 8 µM Ac-DEVD-CHO as indicated and analyzed by SDS-PAGE. Proteins were
visualized by Coomassie Blue staining (lanes 1-6) or by
immunoblotting with an anti-(His)
antibody (lanes
7-10). M, molecular mass markers in kilodaltons. Lane 2 contains unincubated CMH-1 sample. C, samples
from B were diluted 1000-fold to give a CMH-1 concentration of
2 nM and were then incubated for 30 min at 37 °C with in vitro translated
S-labeled PARP in the
presence or absence of Ac-DEVD-CHO as described under ``Materials
and Methods.'' Cleavage products were analyzed by SDS-PAGE and
fluorography. PARP(T) denotes the uncleaved truncated form of
PARP and PARP* the cleavage product. Lane B, buffer
control. D, (His)
-CMH-1 was incubated with
purified granzyme B as in B. Reactions were stopped at the
indicated times and analyzed by Coomassie Blue
staining.
Figure 4:
Cleavage of C186S CMH-1 protein by
granzyme B. Purified (His)-tagged C186S (lanes
2-5 and 8-11) or wild type (lanes 6 and 7) CMH-1 were incubated for 1 h at 37 °C with granzyme B
or buffer in the presence or absence of Ac-DEVD-CHO as in Fig. 3B and then analyzed by SDS-PAGE and Coomassie
Blue staining (lanes 1-7) or by immunoblotting with an
anti-(His)
antibody (lanes 8-11). Lane 1 contains C186S CMH-1 that has not been
incubated.
Figure 5: Cleavage by granzyme B and activity of CMH-1 processing site mutants. A, purified wild type (lanes 7 and 8), D23E (lanes 3-6), or D198E (lanes 10-13) mutant CMH-1 proteins were incubated at 37 °C for 1 h with granzyme B or buffer in the presence or absence of Ac-DEVD-CHO as in Fig. 3B and analyzed by SDS-PAGE and Coomassie Blue staining. Lanes 2 and 9 contain unincubated D23E and D198E samples, respectively. B, samples from A were diluted 1000-fold, and their PARP cleavage activity was determined as described in Fig. 3C.
To determine if granzyme B
cleaves both Asp-Ala
and
Asp
-Ser
of CMH-1 or if CMH-1 activity is
involved in the processing of itself, we included the potent CMH-1
inhibitor Ac-DEVD-CHO in the reaction. In the presence of the
inhibitor, the 5-kDa peptide no longer appeared (Fig. 3B and 4). Instead, a peptide of 25 kDa (p25) became visible and
accumulated over time (Fig. 3, B and D).
Immunoblotting indicated that this peptide contained the (His)
tag (Fig. 3B) suggesting that it is the
unprocessed p5-p20 peptide (Fig. 3A). When we examined
the cleavage products at various time points following granzyme B
addition, we found that in the absence of the inhibitor, p25 initially
appeared and then disappeared (Fig. 3D). The simplest
interpretation of these results is that granzyme B cleaves the
full-length CMH-1 p37 (and p32) between Asp
and
Ser
to form p25 and p12 (p20 and p12), which leads to an
increased autocleavage at Asp
-Ala
of p25 to
form p20 and p5.
To further confirm that cleavage between p20 and p5 is mediated solely by autocatalytic activity of CMH-1, we expressed a mutant CMH-1 in which the putative active site cysteine in the conserved QACRG motif was changed to serine (C186S) and examined its cleavage by granzyme B (Fig. 4). First, we found that the purified C186S CMH-1 protein contained a single major polypeptide of 37 kDa, consistent with p32 in the wild type CMH-1 preparation being an autocatalytic product. Upon incubation of C186S CMH-1 with purified granzyme B, the 37-kDa peptide was cleaved into p25 and p12 with little p20 and p5. These results establish that p20 and p5 are derived from p25 and that this cleavage is mediated solely by autocatalysis. A small amount of an approximately 31-kDa peptide was also present in the C186S CMH-1 preparation and was apparently cleaved into a 19-kDa peptide upon incubation with granzyme B. The nature of this peptide was not further examined.
In contrast, purified D23E protein contained mainly the
37-kDa peptide with a small amount of p25 and p12, consistent with the
notion that the p32 in the wild type CMH-1 preparation is derived from
p37 by autocleavage at Asp. As expected, incubation of
D23E CMH-1 with granzyme B resulted in the cleavage of p37 into p25 and
p12 (Fig. 5A). A small amount of p20 also appeared
after incubation whether granzyme B was present or not, and its
appearance was blocked by Ac-DEVD-CHO, indicating that it is a product
of an autocatalytic event. This may be due to the presence of an
alternative processing site near Asp
, or the autocatalytic
activity of CMH-1 may be capable of cleaving Glu
at the P1
position albeit with lower efficiency.
Finally, we investigated if
cleavage at Asp is required for CMH-1 activity. Purified
wild type or D23E CMH-1 proteins were treated with granzyme B and then
incubated with PARP produced by in vitro translation. As shown
in Fig. 5B, at equal concentrations both the wild type
and D23E CMH-1 cleaved PARP to a similar extent. In addition, an
untreated D23E preparation (Fig. 5A, lane 2)
showed some activity against PARP while the untreated wild type CMH-1
did not. This was consistent with the presence of a small amount of p25
and p12 in the D23E but not in the wild type CMH-1 preparation.
Furthermore, unincubated D23E CMH-1 containing p25 but little p20
cleaved PARP in a similar manner as the preincubated sample, which
contained p25 and p20, suggesting that p25/p12 is as active as the
fully processed p20/p12 protease (compare lanes 2 and 3 in Fig. 5A). These results indicate that
activation of CMH-1 requires cleavage between p20 and p12 at position
Asp
, whereas the cleavage between p20 and p5 is not
required.
CMH-1 was recently identified based on its sequence
homology to the ICE/Ced-3 family of
proteases(13, 14, 15) . It has been shown
that CMH-1 is processed during Fas- and tumor necrosis factor
-induced apoptosis (15) . Although the physiological
function of CMH-1 remains unclear, its ability to cleave PARP and
induce apoptosis suggests that it may play a role in cell death. In
this report, we demonstrate that CMH-1 can be cleaved and activated by
granzyme B in vitro, suggesting that CMH-1, a protease
localized in the cytoplasm(15) , may be involved in a protease
cascade that mediates CTL-induced cell killing in vivo. Our
findings that CMH-1 is cleaved 25-50 times more efficiently than
CPP32 by granzyme B implies that CMH-1 may be a primary target for
granzyme B. Furthermore, the close homology between CMH-1 and CPP32
suggests that the mechanism of granzyme B-mediated CMH-1 activation may
be applicable to CPP32. Our finding that the removal of the prosequence
is not required for CMH-1 activation in vitro points to the
possibility that the prosequence may be involved in other aspects of
CMH-1 regulation in vivo.