From the Department of Biochemistry and Molecular
Biology, Monash University 3800, Melbourne, Victoria, Australia,
§ Biomedicinal Chemistry Group, School of Pharmacy, The
Queen's University of Belfast, Belfast BT9 7BL, United Kingdom, the
¶ Australian Centre for Blood Diseases, Box Hill Hospital, Box
Hill 3128, Melbourne, Victoria, Australia, and the
Baker Medical
Research Institute, St. Kilda Central
8008, Melbourne, Victoria, Australia
Received for publication, July 25, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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The cytotoxic lymphocyte serine
proteinase granzyme B induces apoptosis of abnormal cells by cleaving
intracellular proteins at sites similar to those cleaved by caspases.
Understanding the substrate specificity of granzyme B will help to
identify natural targets and develop better inhibitors or substrates.
Here we have used the interaction of human granzyme B with a cognate
serpin, proteinase inhibitor 9 (PI-9), to examine its substrate
sequence requirements. Cleavage and sequencing experiments demonstrated that Glu340 is the P1 residue in the PI-9 RCL,
consistent with the preference of granzyme B for acidic P1 residues.
Ala-scanning mutagenesis demonstrated that the P4-P4' region of the
PI-9 RCL is important for interaction with granzyme B, and that the P4'
residue (Glu344) is required for efficient
serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9
RCL sequence and containing either P1 Glu or P1 Asp were cleaved by
granzyme B (kcat/Km 9.5 × 103 and 1.2 × 105
s Cytotoxic lymphocytes
(CLs)1 play a key role in
cell-mediated immunity, destroying foreign, virus-infected, or tumor
cells (1). Contact between a CL and a target cell leads to the release of cytotoxins such perforin, serine proteinases (granzymes), and death
ligands (FasL and TRAIL) from specialized CL secretory granules into the intercellular space. Target cell death then proceeds by the
induction of apoptosis using either of two pathways: activation of
death receptors on the target cell surface coupled to caspase activation within the cell or uptake of granzymes in a
perforin-dependent manner that leads to caspase activation
and intracellular proteolysis (reviewed in Refs. 1 and 2).
The molecular basis of granzyme-induced cell death is not fully
understood. The current model is that perforin and granzymes are
endocytosed by the target cell and that perforin eventually disrupts
the endocytic vesicle, thus releasing granzymes into the cytoplasm (3,
4). Caspase activation, loss of mitochondrial membrane potential,
proteolysis of key housekeeping proteins, DNA degradation, and the
disintegration of cellular structures follows rapidly. A key granule
cytotoxin in this process is the serine proteinase, granzyme B. It is
an unusual proteinase with a preference for cleaving after Asp, a
property that it shares with caspases. On entering the cytoplasm of a
cell, granzyme B induces death by at least two pathways. One pathway is
caspase dependent and results in rapid DNA degradation and death. The other pathway is caspase independent and may involve direct
degradation of essential proteins such as DNA-PKcs, NuMA, and
PARP (5). Both pathways involve loss of mitochondrial membrane
potential. The pivotal role of granzyme B in granule-mediated cell
death is illustrated by mice lacking granzyme B. These animals produce CLs that are unable to induce rapid DNA degradation and death of target
cells, although slower killing occurs, possibly mediated by other
granzymes (6).
Because caspases themselves are activated by cleavage after Asp,
granzyme B is able to proteolytically activate several caspases in vitro (7), and there is evidence that this also occurs in cells killed by CLs (8, 9). Of the other known granzyme B substrates
within cells, most appear to be cleaved after Asp but not necessarily
at sites recognized by caspases. Granzyme B has also been implicated in
the cleavage of the BCL-2 family member BID (10), in components of the
DNA repair machinery, and in the generation of autoimmune antigens
(11). Thus, identification of noncaspase substrates and elucidation of
the cleavage specificity of granzyme B is attracting increasing
interest. Screening of combinatorial tetra-peptide substrate libraries
has indicated that granzyme B has an optimal P4-P1 recognition motif of
Ile-Glu-Pro-Asp (12), resembling the activation sites of procaspase 3 and procaspase 7, both of which are activated by granzyme B (7). It
also resembles the optimal recognition motif of the group III or
initiator caspases, implying that granzyme B functions as an initiator
of the caspase proteolytic cascade. This motif has recently been
confirmed using combinatorial methods and extended using phage display
to suggest that the preferred P2' residue is Gly and that the consensus
recognition motif for granzyme B is Ile-Glu-Xaa-(Asp/Xaa)-Gly (13).
However, synthesis of a peptide comprising an optimized P4-P2' sequence resulted in a molecule that was not cleaved efficiently by granzyme B
(13), suggesting that residues outside this motif contribute to
substrate binding.
A different approach to understanding the interaction of granzyme B
with substrates is to study its interaction with a natural inhibitor,
such as a serpin. Serpins belong to a large metazoan and virus protein
superfamily (14) and have a common structure and mode of action; each
contains a variable C-terminal reactive center loop (RCL) resembling
the substrate of its cognate proteinase that is flanked by two highly
conserved hinge domains. On proteinase binding, the serpin is cleaved
and undergoes a conformational change leading to the irreversible
locking of the serpin-proteinase complex. Cleavage of the serpin by the
proteinase occurs between two residues in the loop designated P1 and
P1'. The P1 residue is crucial and largely dictates the specificity of
the serpin-proteinase interaction, whereas residues surrounding the
cleavage site contribute to the affinity of the interaction (15).
The human serpin, proteinase inhibitor 9 (PI-9), is an efficient
inhibitor of granzyme B that prevents granzyme B-mediated apoptosis in
certain cell types and is thought to protect CLs from autolysis (16).
It follows that the PI-9 RCL resembles a natural substrate of granzyme
B and that residues within the RCL important for inhibitory function
interact with the substrate binding pocket of the enzyme. Here we show
by directed mutagenesis that residues within the P4-P4' sequence of the
PI-9 RCL are crucial for inhibitory function. In particular, we
demonstrate that the P3' and P4' residues are important for binding and
that the P4' Glu probably forms a salt bridge with 27Lys of
granzyme B, which indicates that, unlike many other serine proteinases,
granzyme B has extended substrate specificity. Synthesis and analysis
of peptides based on the PI-9 RCL sequence and incorporating P1'-P4'
residues confirms this finding and has enabled the development of
specific and sensitive granzyme B substrates.
Recombinant Protein Production and Mutagenesis--
Recombinant
PI-9 was produced using a Pichia pastoris expression system
and purified as described (16). Recombinant human granzyme B zymogen
was produced and purified using a similar system and then activated by
recombinant bovine enterokinase cleavage (17). Recombinant human
caspases were produced as described (16). Site-directed mutagenesis of
both PI-9 and granzyme B followed the Deng and Nickoloff method (18) as
described in Ref. 16. Sequences of the mutagenic and selection primers
will be supplied by the corresponding author on request.
Protein Analysis--
50 µg of the PI-9 T327R mutant was
incubated with 5 µg of recombinant granzyme B in 125 µl of 20 mM Tris, pH 7.4, 0.15 M NaCl for 30 min at
37 °C. 0.5 µl of Kinetic Analysis--
The interaction of granzyme B with PI-9
and derivatives was analyzed using procedures described in Ref. 19.
Protein concentrations were determined by Bradford assay, and as active
site titrants of granzyme B were not available, protease activity was
routinely determined by assessing complex formation with a 2-fold
excess of highly purified wild type PI-9. Only batches of granzyme B in
which greater than 95% of protease showed complex forming activity with PI-9 were used in the kinetic experiments. Briefly, under pseudo-first order conditions a constant amount of enzyme (0.2 nM) was mixed with different concentrations of inhibitor
and excess substrate (100 µM substrate 2) in 500 µl.
Progress curves were monitored at 37 °C for 2 h in a
Perkin-Elmer LS50B spectrofluorimeter (excitation, 320 nm; emission,
420 nm). The stoichiometry of inhibition (SI) and rate constant
(kass) for the interaction of granzyme B with
PI-9 and each mutant were derived as described (19, 20). Experiments
were performed at least three times, and the weighted means of the
determinations are reported.
Cleavage of quenched fluorescence and spectrophotometric granzyme B
substrates and derivation of kcat values was as
described (17). The substrate IETD-pNA was purchased from Calbiochem. Caspase cleavage of commercial substrates was performed as previously (16) in 100 mM HEPES, pH 7.5, 10% sucrose, 0.5 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol
containing 50 µM of the appropriate substrate for 1 h at 37 °C. An arbitrary activity ratio was used to compare caspase
activity on the various substrates and is defined as maximal
fluorescence in the presence of caspase divided by maximal fluorescence
in the absence of caspase.
Substrate Synthesis--
The various internally quenched
substrates were prepared on a 25-µmol scale on a Rink AM amide
polystyrene-based resin (resin particles are functionalized with
4-[2',4'-dimethoxyphenyl-Fmoc-aminomethyl]-phenoxy linker), using
standard solid phase Fmoc protocols (21) on a Perkin Elmer Biosciences
Synergy instrument. The
When samples of the P1 Asp-containing peptides were analyzed by
reversed-phase HPLC, two major components were detected in every
instance. This is probably due to the formation of Molecular Modeling of the PI-9 RCL-Granzyme B
Interaction--
Human granzyme B is 69% identical to rat granzyme B. The x-ray crystal structure of rat granzyme B complexed to a variant of
the small serine proteinase inhibitor ecotin (Ref. 25; Protein Data
Bank identifier 1FI8) was used as a template to model human granzyme B
using the program MODELLER (26). The structure of rat granzyme B in
complex with ecotin enabled us to model the P7-P1 region of PI-9 into
the active site of human granzyme B using using techniques similar to
those previously described (27). Specifically, we superposed our model
of human granzyme B onto the x-ray crystal structure of rat granzyme
B-ecotin using a program by Arthur Lesk (28). We then removed the
structure of rat granzyme B and residues 6-77 of ecotin (residues
77-84 representing the P7-P1 region) to leave the model of human
granzyme B with the P7-P1 region of ecotin. We converted the P7-P1
sequence SSPIEPD of ecotin to that of PI-9 (SCFVVAE) using the
"mutate" facility in the program Quanta (MSI Inc., San Diego, CA).
Because the P' region of the inhibitor is missing in the structure of
the rat granzyme B-ecotin complex, we used the structure of trypsin
bound to ecotin ((29); Protein Data Bank identifier 1SLU) as a template to build the P1'-P5' of the RCL of PI-9 into our model of human granzyme B. Trypsin and rat granzyme B share 36.5% sequence identity and superpose with a root mean square deviation of 0.95 Angstroms/atom over 210 C Identification of the P1 Residue in the PI-9 RCL--
The identity
of the PI-9 P1 residue as Glu340 has been inferred
previously by serpin sequence comparisons and by the established preference of granzyme B for acidic P1 residues (31, 32). This
assignment is supported by mutagenesis experiments substituting Glu340 with Ala or Asp, which resulted in serpins with
insignificant or very low inhibitory activity against granzyme B (16).
However, sequence comparisons are unreliable for identifying P1
residues because the RCL length varies from serpin to serpin, and
different residues may be utilized in interactions with different
proteinases. The latter point is illustrated by the PI-9-related
serpin, PI-6, which uses different P1 residues for interactions with
chymotrypsin and thrombin (33). In the case of PI-9, there is another
acidic residue (Glu344) in the RCL, and granzyme B also
cleaves after Met (32), so it is formally possible that either
Glu344 or Met343 acts as the P1 residue and
that Glu340 contributes only to initial inhibitor binding.
To confirm the assignment of Glu340 as the P1 residue,
cleavage and peptide sequencing experiments were undertaken. To avoid rapid inhibition of the enzyme by PI-9 and promote cleavage of the
serpin by granzyme B, a variant of PI-9 was used. This carries a
mutation in the proximal hinge (Thr326 to Arg) previously
shown to abrogate inhibitory activity and convert the serpin into a
granzyme B substrate (16). The recombinant serpin was incubated with
native granzyme B, the resulting 38- and 4-kDa cleavage products were
alkylated and separated by HPLC (Fig. 1),
and then the smaller fragment was subjected to amino acid sequence
analysis (data not shown). Because of low level sequence contamination,
the residues in the first and fifth positions were equivocal, but the
sequence amino terminus-Xaa-Cys-Met-Glu-Xaa-Gly-Pro-Arg was
otherwise unambiguous and corresponded to the PI-9 RCL sequence from
Cys341 to Arg348. This indicated that granzyme
B cleavage had occurred between Glu340 and
Cys341, proving that for this protease Glu340
is the P1 residue in the PI-9 RCL.
Scanning Mutagenesis of the PI-9 RCL--
The serpin-proteinase
interaction involves the reversible formation of an initial complex,
followed by conversion to a locked complex by cleavage of the serpin
between the P1 and P1' residues, which triggers rapid insertion of the
RCL into the serpin A
It is generally accepted that the specificity and affinity of the
serpin-proteinase interaction is dictated primarily by the P1 residue
(39). This is consistent with the inhibition of degradative proteinases
that have a broad substrate range and high catalytic rate (pancreatic,
neutrophil, and lysosomal proteinases) and do not have stringent
sequence motifs around the P1 that influence substrate binding. It is
also classically illustrated by the Pittsburgh mutation of
We have previously shown that alteration of the PI-9 P1 residue to Asp
results in an inhibitor that is 100-fold less effective and has
properties of a granzyme B substrate (Fig.
2 and Ref. 16). We have also shown that
the PI-9 granzyme B interaction requires a mobile RCL, because mutation
of the proximal hinge abrogates inhibitory activity and converts PI-9
into a substrate (Fig. 2). To identify other residues in the PI-9 RCL
loop important for granzyme B inhibitory function, we carried out
site-directed mutagenesis, substituting the P7 to P5' residues in turn
with Ala (except the P2 Ala, which was replaced with Gly). Mutated serpins were produced in the P. pastoris system and prepared
to greater than 90% purity using immobilized metal affinity
chromatography (for example, see Fig. 2).
Mutants were tested for the ability to form SDS-stable complexes with
granzyme B (illustrated in Fig. 2) and then were subjected to kinetic
analysis. SI and kass values were derived for
the interaction of each serpin with granzyme B. With the exception of
the variant with a mutation in the proximal hinge, all mutants retained
inhibitory activity as indicated by their ability to complex with
granzyme B (data not shown), but there were marked differences noted in the stoichiometry and kinetics of the interactions. As shown in Table
I and discussed below, pronounced effects
on the inhibitory capacity of PI-9 were observed for mutations at P7,
P4, P3, P1, P3', and P4'.
Properties of the P7-P1 Mutants--
Molecular modeling shows that
P7 probably does not interact with the active site of granzyme B (see
below). Mutating this residue increased the SI of the PI-9-granzyme B
interaction and decreased the kass 3-fold,
indicating that inhibitory function is affected. Because the SI × kass value for the interaction is close to that
of wild type, indicating little effect on intermediate formation, it is
unlikely that this mutation affects proteinase docking. It is more
likely that it slows or prevents complete insertion of the RCL into the
A
By contrast, mutating the PI-9 P4-P1 residues apparently affects
proteinase binding to the serpin prior to RCL insertion, and the
results are consistent with the known substrate characteristics and
preferences of granzyme B (12, 13). Combinatorial and deletion analysis
has shown that a minimal peptide substrate for granzyme B must contain
a P4 residue and that the optimal sequence is Ile-Glu-(Pro/Thr)-Asp
(12). However, the relative contribution of each residue in this
sequence to substrate binding has not been determined. Granzyme B will
cleave peptide substrates having either Ile or Val at the P4 position
(although Ile is slightly preferred), and most natural substrates have
either P4 Ile or Val (see Table III). Thus, the presence of a P4 Val in
the wild type PI-9 RCL is consistent with this residue playing an
important role in granzyme B binding. Our analysis showed that
substitution of the P4 Val for Ala in the PI-9 RCL results in decreased
inhibitory function as indicated by a 4-fold decrease in the
kass, emphasizing the importance of Val in this
position for the binding of granzyme B to substrates and inhibitors.
Peptide substrate analysis has indicated a preference for Glu at P3
(12, 13), but examination of natural substrates show that human
granzyme B tolerates other residues at this position (Table III). At
present it is not clear how important the P3 residue is for granzyme B
binding. Tetrapeptide substrates with Val at this position are cleaved
by rat granzyme B at 20% of the efficiency of substrates
containing Glu (13), whereas substrates containing P3 Ala are cleaved
at 60% efficiency. Interestingly human granzyme B does not cleave
substrates containing P3 Ala or Val as efficiently (<20%) as rat
granzyme B (12, 13), indicating subtle differences in the substrate
binding characteristics of granzyme B from different species. Our
analysis shows that converting the PI-9 P3 from Val to Ala has a
pronounced negative effect on inhibition of human granzyme B
(approximately a 10-fold decrease in the kass)
without markedly affecting the SI, which illustrates that P3 is
important for enzyme binding and suggests that it may be more important than the P4 residue.
At P2 there is reported to be a slight preference for proline (12, 13),
but substrates containing Ala or Gly are also cleaved. Interestingly,
peptide substrates with P2 Gly are cleaved less efficiently than those
containing P2 Ala by both rat and human granzyme B, and this is
reflected in the poorer inhibitory capacity we observed for the PI-9
mutant carrying a P2 Ala to Gly substitution (Table I). Nevertheless,
our data support the previous conclusion that the P2 residue does not
contribute significantly to substrate binding.
Finally, it is known that Asp is preferred at P1 for substrate cleavage
by granzyme B (32, 41). As we have previously shown (illustrated in
Fig. 2 and Table I), substitution of the P1 Glu with Asp results in a
much poorer inhibitor that is cleaved efficiently by granzyme B (16).
Substitution with Ala resulted in an essentially inactive inhibitor,
confirming the importance of the P1 in substrate and inhibitory
function. Overall, our results clearly show that efficient substrate or
inhibitor binding to human granzyme B requires an extended sequence
upstream of the cleavage site and that the relative importance of the
various residues is P1 > P3 > P4 > P2 > P5.
Properties of the P' Mutants--
Substrate phage display analysis
has suggested that unlike caspases, granzyme B cleavage efficiency is
influenced by P' residues in the substrate and that Gly is preferred at
P2' (13). Gly is found at P2' in a number of natural granzyme B
substrates (Table III). However, a model peptide
Ac-Ile-Glu-Pro-Asp-Trp-Gly-Ala-NH2 comprising the optimal
P4-P1 residues identified via combinatorial analysis, and the optimal
P1' and P2' residues identified by phage display was not cleaved
efficiently by granzyme B (13). In fact, it was a far worse substrate
than a tetrapeptide comprising Ile-Glu-Pro-Asp, suggesting that at
least in this context the P' residues negatively influence the granzyme
B-substrate interaction and that a Trp-Gly pair is suboptimal.
Our analysis showed that mutation of the PI-9 P1' and P2' residues to
Ala does not adversely affect inhibitory function (Table I) nor does
the conservative substitution of Cys to Ser at the P1' position. This
suggests that the P1' and P2' residues are not crucial for the
interaction of granzyme B with longer authentic substrate sequences. By
contrast, mutation of the P3' residue mildly affected inhibitory
function (to approximately the same degree as the P4 mutation), and
examination of the data suggests that proteinase binding rather than
RCL insertion is affected. Substitution of the P4' residue had a most
pronounced effect, doubling the SI and lowering the
kass by over 10-fold. The properties of this
mutant suggest that in the absence of an acidic P4' residue more serpin
is required for inhibition because it binds far less effectively to the
enzyme. This implies that substrates without an acidic P4' residue will
be cleaved less efficiently by granzyme B.
To test whether a negatively charged residue is important at the P4'
position, we replaced Glu344 with Asp. The resulting
protein was a more effective inhibitor of granzyme B than the the P4'
Ala mutant (Table I) but had perturbed characteristics compared with
wild type, indicating that granzyme B binding is not as tight. Thus,
efficient interaction of human granzyme B with substrates or inhibitors
probably requires an acidic residue at P4', preferably Glu. This is
supported by the fact that the two other serpins known to interact with
human granzyme B in vitro, SPI6 and CrmA (42, 43), do not
have acidic P4' residues and exhibit kinetic characteristics very
similar to the PI9 P4' Ala mutant.
Synthesis of Granzyme B Substrates Based on the PI-9 RCL--
To
further test the idea that the P4-P4' region of PI-9 mimicks a
substrate of granzyme B, we synthesized quenched fluorescence substrates based on this sequence. In the first instance we synthesized two peptides: abz-Val-Val-Ala-Glu-Ser-Ser-Met-Glu-Lys-dnp (substrate 1)
and abz-Val-Val-Ala-Asp-Ser-Ser-Met-Glu-Lys-dnp (substrate 2). For ease
of synthesis and peptide stability, we used Ser at the P1' and P2'
positions instead of Cys. This was suggested by mutagenesis experiments
demonstrating that substitution of the P1' Cys with either Ser or Ala
had no significant effect on inhibitory function (Table I).
The only difference between substrates 1 and 2 is the identity of the
P1 residue. Given the preference of granzyme B for Asp, and the fact
that the PI-9 E340D mutation resulted in a molecule with
characteristics of a substrate rather than inhibitor (Table I), we
predicted that substrate 2 would be cleaved more efficiently. As shown
in Table II, both substrates were cleaved
by granzyme B, but substrate 2 was cleaved three times more efficiently
than substrate 1. The kinetics compared favorably with published values for the cleavage of tetrapeptides by granzyme B; the best tetrapeptide (Ac-Ile-Glu-Pro-Asp-p-nitroanilide (Ref1 in Table II))
yields a Km of 57 µM and
kcat of 4.16 s
Although substrate 2 binds more tightly to granzyme B than the best
tetrapeptide substrate, it is cleaved at a slower rate. This lower
cleavage efficiency may be due to a suboptimal P4-P1 sequence. To test
this idea, we synthesized an idealized granzyme B substrate comprising
the best reported P4-P1 sequence linked to the P1'-P4' sequence of PI-9
(substrate 3). This substrate bound 10 times more tightly to granzyme B
than the tetrapeptide substrate and was turned over at an equivalent
rate, yielding a 11-fold higher specificity constant (Table II).
Interestingly, the sequence of substrate 3 resembles the known
activation site for granzyme B on caspase 3 (Table
III), which includes an acidic P4'
residue and is a known in vivo target of granzyme B (8, 44).
To test the importance of the P4' residue in the interaction between
granzyme B and substrates, we also synthesized a substrate identical to
substrate 2 except that the P4' Glu is replaced by Ala (substrate 4).
As shown in Table II, substrate 4 did not bind as tightly as substrate
2 to granzyme B (5-fold increase in Km) and was
turned over more efficiently (2.5-fold increase in
kcat). These results are consistent with an
important role for the P4' residue in a direct interaction with
granzyme B rather than in maintenance of PI-9 RCL conformation.
Modeling of the Granzyme B-PI-9 RCL Interaction--
To put our
results in a structural context, we modeled human granzyme B using the
structure of rat granzyme B (25) as a template (the two proteins are
69% identical). We then docked the P7-P5' portion of the PI-9 RCL into
the active site of the model using the three-dimensional structures of
a rat granzyme B-ecotin complex (25) and a trypsin-ecotin complex (29)
as guides.
As shown in Fig. 3, the catalytic site of
human granzyme B is predicted to be part of a pronounced cleft that
would allow the binding of an extended substrate sequence and
accommodate the P6-P4' residues of PI-9. This is consistent with our
observations that the P4-P4' residues can influence substrate and
inhibitor binding. However, the most striking result of the modeling
was that the P4' Glu of the PI-9 RCL is predicted to be in close
proximity to Lys27 of mature granzyme B, and the two
residues are likely to form a salt bridge. Specifically, in the side
chain conformations of the model the Site-directed Mutagenesis of Granzyme B--
To test the idea that
Lys27 in granzyme B interacts with the P4' Glu of PI-9, we
produced two mutants of granzyme B: Lys27 to Ala, and
Lys24 to Ala. We predicted that the first mutant would bind
less efficiently to PI-9 and to substrates containing P' residues
because the salt bridge can no longer form. By contrast, the second
mutant should bind as efficiently wild type granzyme B to inhibitors or
substrates because Lys24 is not predicted to participate in
intermolecular interactions.
As shown in Table IV, both mutants
cleaved a tetrapeptide substrate lacking P' residues to the same extent
as wild type, which demonstrates that neither mutation perturbs the
catalytic function of the enzyme. By contrast, when compared with wild
type or the K24A mutant, the K27A mutant showed a marked decrease in
the ability to bind and cleave a substrate (substrate 3) containing P'
residues. The decrease in kcat for the
interaction suggests that the P' residues play a role in positioning
the substrate in the active site cleft. Furthermore, the K27A mutant
showed a marked decrease in its ability to interact with PI-9 (a 4-fold
increase in the SI and a 20-fold decrease in
kass). Taken together, these results strongly
support the prediction that the P4' Glu of PI-9 is involved in a salt
bridge with Lys27 of granzyme B and suggest that high
affinity interactions of granzyme B with substrates and inhibitors
require the participation of P' residues particularly an acidic
P4'.
Caspase Recognition of the Granzyme B Substrates--
Caspases and
granzyme B show similarities in substrate recognition and biological
function. Combinatorial analyses suggest that granzyme B and the group
III (activator) caspases prefer substrates with similar P4-P1
sequences, consistent with their roles as upstream activators of the
caspase cascade and apoptosis (12). To see how far this similarity
extends, we tested the ability of three group III caspases (caspases 6, 8, and 10) to cleave our substrates 1-3 and compared this to caspase 5 (group I) and caspase 3 (group II). As shown in Fig.
4, none of the caspases effectively
cleaved substrates 1 or 2, whereas two of the group III caspases
(caspase 6 and 10) cleaved substrate 3. The failure of caspases to
cleave substrates 1 and 2 probably reflect stricter P4-P1 requirements
compared with granzyme B. For example, group I caspases prefer His at
P2 (and caspase 5 also prefers Glu at P3); group II caspases prefer Asp
at P4; and group III caspases prefer Glu at P2 (12). The ability of
caspase 6 and caspase 10 to cleave substrate 3 is not surprising, given
that its P4-P1 sequence is close to optimal. However, the failure of
caspase 8 to cleave substrate 4 is unexpected and indicates that the P' residues in this substrate prevent efficient interaction with the
binding site of the enzyme. Taken together these results show that
substrates based on the PI-9 RCL (substrates 1 and 2) can be used to
discriminate caspases and granzyme B in situations where both are
active.
The Role of the P' Residues in Substrate and Inhibitor
Binding--
Overall, our results indicate that the P1'-P4' residues
of granzyme B substrates are important for substrate binding, but the
optimal P1'-P4' sequence for granzyme B recognition remains to be
determined, perhaps by combinatorial or phage display methods. Our work
also suggests that efficient and selective granzyme B inhibitors
require P' residues and may explain why effective tetrapeptide or other
synthetic granzyme B inhibitors have not been reported to date
(45).
An acidic P4' residue in the PI-9 RCL is consistent with the idea that
this inhibitor has evolved to maximize granzyme B binding at the
expense of cleavage, a point that is reinforced by the presence of the
nonfavored Glu at the P1 position. Examination of the known granzyme B
cleavage sites on protein substrates reveals that some like those in
caspase 3 and PARP include acidic P4' residues (Table III).
However, many others do not have acidic P4' residues in their
characterized granzyme B cleavage sites. This suggests that granzyme B
has two substrate classes that are defined by the presence or absence
of acidic P4' residues: one set that it binds with high affinity but
cleaves slowly, and another set that it binds with lower affinity but
cleaves more rapidly. It also implies a hierarchy of targets for
granzyme B in the cell, which is consistent with its role as an
initiator of apoptosis; it binds preferentially to and inactivates
proteins that act early in apoptotic pathways and then less selectively
degrades a secondary set of targets. However, it is not possible to
group known natural substrates into subcategories to predict which are
early (low Km, low kcat) or
late (higher Km and kcat)
targets because binding and catalytic constants have not been reported for their interactions with granzyme B (Table III).
In closing, our results demonstrate that P4-P4' sequences in natural
substrates and inhibitors are required for efficient binding to human
granzyme B and that the highest affinity is observed when an acidic P4'
residue is present. This is consonant with the narrow substrate range
of granzyme B and the regulatory role it plays in cytotoxic
lymphocyte-mediated apoptosis, in contrast to the degradative serine
proteinases that have a large range of substrates and low sequence
selectivity. It is interesting to note that so far only one other
serine proteinase has been shown to have such an extended sequence
requirement for substrate or inhibitor binding. Interaction of
tissue-type plasminogen activator with its cognate inhibitor
plasminogen activator inhibitor 1 has been shown to require the P1'-P5'
residues of the serpin (46). This is entirely consistent with the
regulatory role of tPA in plasminogen activation and its restricted
range of substrates.
1 M
1, respectively) but were
not recognized by caspases. A substrate containing P1 Asp but lacking
P4' Glu was cleaved less efficiently (kcat/Km 5.3 × 104 s
1 M
1). An
idealized substrate comprising the previously described optimal P4-P1
sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was
efficiently cleaved by granzyme B
(kcat/Km 7.5 × 105 s
1 M
1) and was
also recognized by caspases. This contrasts with the literature value
for a tetrapeptide comprising the same P4-P1 sequence
(kcat/Km 6.7 × 104 s
1 M
1) and
confirms that P' residues promote efficient interaction of granzyme B
with substrates. Finally, molecular modeling predicted that PI-9
Glu344 forms a salt bridge with Lys27 of
granzyme B, and we showed that a K27A mutant of granzyme B binds
less efficiently to PI-9 and to substrates containing a P4' Glu. We
conclude that granzyme B requires an extended substrate sequence for
specific and efficient binding and propose that an acidic P4' substrate
residue allows discrimination between early (high affinity) and late
(lower affinity) targets during the induction of apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-mercaptoethanol was added, the incubation was
continued for 1 h, and then 0.5 µl of 4-vinyl pyridine was added
for 1 h. The alkylated protein sample was subjected to HPLC
analysis on a Hewlett Packard HP 1090 liquid chromatograph fitted with
a C-18 ODS poly LC column (1 × 100 mm), and data analysis was
performed on a HP 79994A HPLC work station. Diode array detection allowed spectral analysis of the column effluent at 214, 254, and 280 nm. Buffer A comprised 0.1% trifluoroacetic acid (v/v), and buffer B
comprised 0.1% trifluoroacetic acid, 70% CH3CN (v/v). The
column was eluted with a gradient of 0-100% buffer B over 60 min at
50 µl/min. UV absorbing peaks were collected and subjected to 11 cycles of N-terminal amino acid sequencing on a PE-ABI Procise automated peptide sequencer.
-dinitrophenyl (dnp) lysine residue was
introduced into the sequence as its N-
-Fmoc derivative,
employing
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and 1-hydroxybenotriazole activation, in the presence of di-isopropylethylamine, essentially according to the method
of Knorr et al. (22). In contrast, the aminobenzoyl (abz) residue was introduced as its t-butoxycarbonyl-protected
pentafluorophenyl ester (23). On completion of the synthesis, the
peptides were obtained as their C-terminal amides by cleavage from the
support by treatment with a solution of trifluoroacetic acid in 50%
(v/v) dichloromethane containing a carbocation scavenger mixture
(ethanedithiol, water, thioanisole 1:1:2).
-aspartyl-linked sequences arising out of a transpeptidation reaction of the Asp-Ser peptide bond, which is a common problem in the synthesis of such peptides (24). However, the reaction can be reversed by treatment with
dilute ammonia. When applied to our peptides, this protocol resulted in
the generation of products containing one major component. The purity
and identity of each peptide were confirmed using capillary electrophoresis and matrix-assisted laser desorption
ionization-time of flight mass spectroscopy.
atoms (93% of the structure). Superposition of the rat
granzyme B-ecotin complex reveals that the backbone atoms of the P1-P4
residues of ecotin in each structure overlay almost exactly (within
experimental error), consistent with the canonical structure of the
P4-P4' region of the inhibitory loop of small serine proteinase
inhibitors (30). Thus, the trypsin-ecotin structure is a suitable
template for modeling the P' region of the PI-9 RCL in the active site
of human granzyme B. We superposed our model of human granzyme B in
complex with the P7-P1 region of PI-9 onto the trypsin-ecotin complex
using a program by Arthur Lesk (28). We removed the structure of
trypsin and the ecotin molecule with the exception of the P1'-P5'
region. We then converted the P1'-P5' sequence of ecotin (MHCPD) to
that of PI-9 (CCMES). We created a peptide bond between the P1-P1'
residues and then subjected the entire model of human granzyme B in
complex with the P7-P5' residues of human PI-9 to CHARMm minimization,
first with the backbone atoms of the RCL peptide constrained and later with no constraints. In each case CHARMm minimization was performed to convergence.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (37K):
[in a new window]
Fig. 1.
Identification of the PI-9 P1 residue.
50 µg of the PI-9 T327R (TR) mutant was incubated with 5 µg of recombinant granzyme B (graB), and the reaction
products were alkylated. A, 1% of the sample was analyzed
by 12.5-15% gradient SDS-polyacrylamide gel electrophoresis followed
by silver staining. The 38-kDa N-terminal peptide (N) and
4-kDa C-terminal peptide (C) are indicated. B,
The remaining material was subjected to purification by HPLC, and a
representative elution profile monitored at 214 nm is shown here
(vertical axis is in arbitrary absorbance units
(AU)). The arrowed peak was collected in five
fractions at 56, 58, 59, 60, and 62 min, and the indicated sequence was
obtained from the fraction corresponding to 59 min.
-sheet before deacylation is completed (15,
34, 35). Distortion, inactivation, and translocation of the trapped
proteinase to the opposite pole of the serpin results from RCL
insertion (36). Alternatively, catalysis is completed before RCL
insertion, the proteinase escapes, and the complex dissociates,
releasing cleaved serpin and active enzyme (34). In other words,
following initial complex formation the pathway bifurcates and can
follow an inhibitory or substrate route depending on the rate of RCL
insertion. Kinetic analysis of the interaction can indicate whether
formation of the locked complex is favored (the inhibitory pathway) or
whether cleavage and dissociation is favored (the substrate pathway). For example, if the SI is unitary the inhibitory pathway is favored, whereas higher SI values indicate that the serpin is seen more as a
substrate by the enzyme. The second order association constant (kass) is a measure of the rate of RCL insertion
and complex formation. The higher the kass, the
faster the reaction and the less likely it is to follow the substrate
pathway. Multiplication of the kass by the SI
yields a value for the rate of formation of the nonreversible intermediate that precedes the inhibitory/substrate pathway branch point (37, 38).
1-antitrypsin, which retargets the serpin from elastase to thrombin by conversion of its P1 residue from Met to Arg (39, 40).
However, granzyme B differs from the degradative proteinases in that
its catalytic and biological properties suggest that it is a regulatory
enzyme that acts on a limited set of proteins. In other words, it is
highly selective, and it therefore follows that substrate specificity
is also likely to be influenced by residues around the P1. This is
supported by work showing that the P4 and P3 residues are important in
granzyme B substrates (12) and that, unlike pancreatic serine
proteinases, it is not capable of hydrolyzing amide substrates of less
than three residues (13). Thus, binding and inhibition of granzyme B by
PI-9 is likely to depend on the P4 and P3 residues and probably others surrounding the P1.
View larger version (23K):
[in a new window]
Fig. 2.
Interaction of granzyme B with mutant
serpins. 2-3 µg of recombinant serpin was incubated with 1 µg
of recombinant granzyme B (graB) for 1 h at 37 °C.
Samples were analyzed by 12.5% SDS-polyacrylamide gel electrophoresis
followed by Coomassie Blue staining. Represented are wild type PI-9,
the derivative containing the P1 Glu to Asp substitution
(ED), and the derivative containing the P14 Thr to Arg
substitution (TR). The SDS-stable complex is indicated
(C), as well as cleavage products (CP).
Directed mutagenesis of the PI-9 RCL
-sheet, increasing the probability that the reaction will follow
the substrate pathway.
1 (13), compared
with 13 µM and 1.5 s
1, respectively, for
substrate 2. In terms of the specificity constant (Km/kcat) substrate 2 is
1.8-fold better than the reference substrate.
Synthetic granzyme B substrates based on PI-9
Natural substrates of granzyme B
indicates a cleavage site.
-nitrogen of the lysine is
predicted to form a salt bridge with the glutamic acid residue; it is
2.5 and 2.6 Angstroms from the O
2 and O
1 atoms, respectively.
This would have the effect of stabilizing the protease-RCL binding and
explains why the mutation of the P4' residue to Ala results in a
significantly lower kass. The negative effect of
having Asp at P4' is explained by its shorter side chain, which makes a
salt bridge with Lys27 harder to form. Interestingly, there
is a Lys in the corresponding position in rat but not mouse granzyme B,
suggesting there may be differences in the substrate profiles and
binding characteristics of the granzymes from various species. We also
noted that Lys24 in granzyme B is close to the P4' Glu (5.5 Å) but is too distant to form a salt bridge.
View larger version (71K):
[in a new window]
Fig. 3.
Model of the complex between human granzyme B
and the PI-9 RCL. A, a stereo view of the region around
the active site of human granzyme B (gray) and the RCL of
PI-9 (cyan). The PI-9 P4 residue Glu344 (labeled
red ball and stick) makes a salt bridge
(dashed line) with Lys27 of granzyme B
(dark blue ball and stick). The PI-9 P1 residue
(Glu340) is labeled and shown in magenta ball and
stick. The granzyme B catalytic triad (His44,
Asp88, and 183Ser) is in green ball and
stick. The distance between the O 2 of P4 Glu and the N
of
Lys 27 is 2.6 Angstroms, the angle C
-N
-O
2 is 145° and the
angle N
-O
2-C
is 98.8°°. Distance and angle measurements
were performed using Quanta (MSI Inc.). B, details of the
interaction between the PI-9 RCL peptide and human granzyme B. The
peptide backbone of the PI-9 RCL is shown in cyan, the P4
residue (Glu344) in red stick, and the P1
residue (Glu341) is in magenta stick. Other side
chains are shown in light gray stick. The C
trace of the
proteinase is in black, and Lys27 is shown in
dark blue stick. The active site triad (His44,
Asp88, and Ser183) is in green
stick. Additional side chains and backbone atoms that form
hydrogen bonds (dashed lines) to the RCL of PI-9 are shown
in black and are labeled. For clarity, hydrophobic contacts
are not shown. The figures were produced using MOLSCRIPT (47).
Characteristics of granzyme B mutants
View larger version (29K):
[in a new window]
Fig. 4.
Interaction of caspases with granzyme B
substrates based on PI-9. Similar amounts of Escherichia
coli lysates containing recombinant caspases were incubated with
50 µM of the indicated substrates: z-YVAD-AFC (YVAD),
z-DEVD-AFC (DEVD), substrate 1 (VVAE), substrate 2 (VVAD), and
substrate #3 (IEPD). Source and use of YVAD and DEVD is described in
Ref. 16, and substrates 1-3 are described in Table II. The activity
ratio is defined as the fluorescence intensity of the lysate divided by
the fluorescence intensity of a similar lysate not expressing the
caspase.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. J. Trapani (Peter MacCallum Cancer Institute, Melbourne, Australia) for supplying native granzyme B used in the preliminary stages of the work and Dr. S. Bottomley and Dr. R. Pike for discussions and critical review of the manuscript. We are also grateful to C. Bird for assistance with the figures and to Dr. S. Stone for initial advice and suggesting a Melbourne-Belfast collaboration.
![]() |
FOOTNOTES |
---|
* The work was supported by grants from the National Health and Medical Research Council of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 61-3-9905-3771; Fax: 61-3-9905-4699; E-mail: Phil.Bird@med.monash.edu.au.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M006645200
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ABBREVIATIONS |
---|
The abbreviations used are:
CL, cyotoxic
lymphocyte;
RCL, reactive center loop;
PI-9, proteinase inhibitor 9;
HPLC, high pressure liquid chromatography;
SI, stoichiometry of
inhibition;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
Fmoc, N-(9-fluorenyl)methoxycarbonyl;
dnp, -dinitrophenyl;
abz, aminobenzoyl.
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