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J Biol Chem, Vol. 274, Issue 43, 30468-30473, October 22, 1999
From the Serine proteases (granzymes) contained within the
cytoplasmic granules of cytotoxic T cells and natural killer cells play a variety of roles including the induction of target cell apoptosis, breakdown of extracellular matrix proteins and induction of cytokine secretion by bystander leukocytes. Different granzymes display proteolytic specificities that mimic the activities of trypsin or
chymotrypsin, or may cleave substrates at acidic ("Asp-ase") or at
long unbranched amino acids such as Met ("Met-ase"). Here, we
report that recombinant granzyme H has chymotrypsin-like (chymase) activity, the first report of a human granzyme with this proteolytic specificity. Recombinant 32-kDa granzyme H expressed in the baculovirus vector pBacPAK8 was secreted from Sf21 cells and recovered by Ni-affinity chromatography, using a poly-His tag encoded at the predicted carboxyl terminus of full-length granzyme H cDNA. The granzyme H efficiently cleaved Suc-Phe-Leu-Phe-SBzl (v = 185 nM/s at [S] = 0.217 mM) and also
hydrolyzed Boc-Ala-Ala-X-SBzl (X = Phe,
Tyr, Met, Nle, or Nva) with slower rates but had little tryptase or
Asp-ase activity. Enzymatic activity was inhibited completely by 0.1 mM 3,4-dichloroisocoumarin and 84% by 1.0 mM
phenylmethylsulfonyl fluoride. Fluoresceinated granzyme H was
internalized in a temperature-dependent manner by
Jurkat cells into endosome-like vesicles, suggesting that it can
bind to cell surface receptors similar to those that bind granzyme B. This suggests a hitherto unsuspected intracellular function for
granzyme H.
Granzymes are serine proteases expressed exclusively by cytotoxic
T lymphocytes (CTL)1 and
natural killer (NK) cells, and stored with a pore-forming protein,
perforin, in lysosome-like secretory granules (1). Humans express five
granzymes: granzyme B (grB) which cleaves after Asp residues (Asp-ase)
(2, 3); grA and tryptase-2 which are trypsin-like (cleavage after basic
residues) (4); grM which cleaves after Met and other long, unbranched
hydrophobic residues and is expressed only in NK cells (5); and grH,
whose substrate specificity is unknown (6, 7). Because of its ability
to activate pro-apoptotic caspases and mimic the cleavage of their
downstream substrates, grB has been strongly implicated in inducing
perforin-dependent target cell apoptosis (8, 9), but this
function can also be carried out with lesser efficiency by grA and
tryptase-2 (10). Putative nonapoptotic functions have been described
for grA, including B lymphocyte mitogenesis, thrombin activation,
induction of cytokine secretion by monocytes, and cleavage of
extracellular matrix proteins (proteoglycans, type IV collagens, lamin,
and fibronectin), thus potentially facilitating T and NK cell migration
through the subendothelial matrix (4, 11-13).
Mice express at least nine granzymes, several of which including
granzymes D, E, F, G, and possibly grC display chymotrypsin-like (chymase) activity. This type of proteolytic activity has been postulated to be important for enhancing the membranolytic properties of perforin (14), although this view has remained controversial. Human
CTL/NK cell granules have high levels of chymase activity, but the
enzyme responsible for this activity has yet to be identified, and to
date, no human granzyme with chymase activity has been found (1). As
grH has no direct rodent counterpart and is the only human granzyme
whose enzyme activity remains unknown, it has been postulated to be
responsible for the chymase activity seen in human granule extracts.
GrH is a close structural relative of grB, and shares 71% amino acid
identity with it (6). The genes encoding the two molecules map within
30 kilobases on chromosome 14, and form part of the serine protease
gene cluster that also contains a number of cathepsin genes expressed
in mast cells and myeloid cells (15). Indeed, it has been proposed that
interlocus recombination between the ancestral grB and grH genes led to
substitution of exon 3, intron 3 and part of exon 4 in grH by grB
sequences (16). GrB is expressed by both CTL and NK cells, but it has recently been demonstrated that the 5' noncoding regions of the grH
gene can confer NK-specific expression of a reporter gene expressed in
mice (17). The high degree of conservation between grB and grH has made
detecting and purifying these two granzymes and production of
monospecific reagents problematic, highlighting the need for caution
when interpreting results obtained with antibody or even nucleic acid
probes (18). Thus, it still remains to be determined whether grH is
expressed solely in human NK cells or in a broader array of lymphocytes.
To clarify the many issues of granzyme structure and biological
function raised above, we found it desirable to produce recombinant, enzymatically active grH. Here we report that grH expressed in, and
purified following baculovirus expression in Sf21 insect cells has strong chymase but no tryptase or Asp-ase activity. However, like
grB, it is capable of uptake into potential target cells in
endosome-like vesicles.
Cell Culture--
The human T cell leukemia cell line Jurkat and
the NK leukemia cell line YT were cultured in RPMI medium supplemented
with 10% bovine serum at 37 °C in air containing 5% carbon
dioxide. Insect serum-free adapted Sf21 cells were cultured in
Sf-900 II medium (Life Technologies, Inc.) at 27 °C in room air,
either in static culture or in 1 liter shaker flasks.
Expression and Purification of Recombinant grH--
The cDNA
encoding the full-length mature wild type grH protein was spliced by
overlap extension polymerase chain reaction onto a heterologous leader
sequence known to support secretion of recombinant product when
expressed in serum-free adapted Sf21 cells. The two codons
encoding the activation dipeptide present at the amino terminus of all
granzymes were deleted to facilitate proteolytic activity of the
secreted product (19). A mutated form of the protein was also
engineered by introducing a mutation that altered the active site Ser
residue to Ala. A hexa-His "tag" was also encoded at the predicted
carboxyl terminus of both polypeptides for ease of purification. Both
cDNA constructs were inserted into the EcoRI sites of
pBacPAK8 vector (CLONTECH) and used to infect Sf21 cells according to standard protocols. Final optimized
expression involved infection of Sf21 cells at a multiplicity of
infection of 0.1 in 100 ml of culture medium 3 days prior to harvest.
Culture supernatant was cleared of cellular debris by centrifugation
and then recombinant protein was purified by Ni-affinity
chromatography. The nickel resin (1-ml bed volume per 200-ml culture
supernatant) with bound product was washed extensively in buffer
containing 20 mM imidazole, and then serial 0.5-ml elutions
were performed using an imidazole step gradient commencing at 100 mM. Recombinant product reproducibly eluted at 250 mM imidazole. The grH was then dialyzed against
phosphate-buffered saline or TE (150 mM NaCl, 1 mM EDTA) and stored at 4 °C or Enzyme Assays--
The enzymatic hydrolysis of peptide thioester
substrates was measured in 0.1 M HEPES, 0.5 M
NaCl, pH 7.5 buffer containing 9% Me2SO and at 23 °C in
the presence of DTNB (3). The initial rates were measured at 405 nm
(
Percent inhibition was measured by incubating the enzyme with an
inhibitor (1 or 10 mM) in a buffer for 10 or 20 min at
23 °C. The substrate, DTNB, and buffer were added to assay the
residual enzyme activity using a microplate reader. The substrate
Suc-Phe-Leu-Phe-SBzl was purchased from Enzyme Systems Products,
Livermore, CA. Other substrates and various inhibitors were synthesized
in the Powers Laboratory at the Georgia Institute of Technology, School
of Chemistry and Biochemistry.
Western Blotting--
A polyclonal antiserum raised in rabbits
against human grB and found to cross-react with grH was used to detect
grH and grB in immunoblots, which were performed from 10 or 12.5%
SDS-PAGE gels using an electroblotter (Bio-Rad) and standard protocols.
Confocal Microscopy--
GrB was purified from the lysates of YT
cells as described previously (9). GrH and grB were labeled with
fluorescein isothiocyanate as described previously (9). For each
granzyme, conjugation with fluorescein resulted in the loss of less
than 20% of proteolytic activity. Jurkat cells were centrifuged onto
microscope slides using a cytospin centrifuge and viewed by confocal
microscopy as described (9).
Expression of Recombinant grH in Baculovirus-infected
Cells--
Only in recent times has it become possible to express and
purify granzymes from recombinant sources, thereby facilitating the
study of their biochemical and biological functions. Ley and colleagues
were the first to report the expression of enzymatically active grB in
yeast cells (21), and pro-grA expressed and purified from bacteria then
activated with enterokinase was used by Lieberman and colleagues to
identify potential cell membrane-associated substrates/ligands for this
granzyme (22). Having had no success expressing soluble grH in
bacterial or yeast expression systems, we attempted expression of this
granzyme in baculovirus-infected cells. The full-length cDNA
encoding grH (6) was expressed following deletion of two codons
encoding the amino-terminal activation dipeptide (Glu-Glu) (19), to
allow accurate folding of the nascent polypeptide chain following
processing of the heterologous leader peptide (see "Materials and
Methods"). To assist with assigning the proteolytic specificity of
the recombinant protease, a mutated version of the enzyme in which the
active site Ser182 was replaced with Ala was expressed in
parallel cultures. A hexa-His tag was also engineered at the carboxyl
terminus of both proteins to permit easy purification and
concentration. Following the initial screening of culture supernatants
from a panel of recombinant baculovirus-infected Sf21 cells,
several clones were identified, which secreted 32-kDa proteins
corresponding to Ser-to-Ala mutated or wild type grH, that reacted in
immunoblots with a polyclonal anti-grB antiserum (Fig.
1A). This antiserum was
previously shown to cross-react with epitopes expressed on fusion
proteins of grH with bacterial glutathione S-transferase
(grH-GST), but not grA-GST, or grM-GST (data not shown). Expression
levels of the mutated grH were consistently approximately 1.5-2.5-fold
higher than for wild type enzyme, as estimated by densitometric
measurements of the immunoblot signals (see below). The expressed
proteins consisted of the 26-kDa polypeptide backbone to which ~6 kDa
of mannose-rich carbohydrate had been added, as judged by in
vitro cleavage with endoglycosidase H (data not shown).
GrH Is a Chymase--
Clones expressing the highest amounts
of wild type or mutated grH were selected and expanded in culture, and
the secreted recombinant granzyme protein was purified using
Ni-affinity chromatography. For both the wild type and mutated enzymes,
elution with 250 mM imidazole resulted in highly purified
protein, as judged by SDS-PAGE and silver staining (Fig.
1B). Optimized yields were in the range of 2-4 mg/liter of
harvested supernatant for the mutated grH and typically about one-half
this amount for wild type grH. For example, the final yields obtained
from the four successive 250 mM imidazole fractions shown
in Fig. 1B were 3.8 mg/liter culture supernatant for mutated
enzyme and 1.7 mg/liter for wild type enzyme (Table I). Sequencing of the amino termini of
both proteins indicated efficient removal of the leader peptides, with
>90% of the final product judged to have the predicted mature amino
terminus of grH (Ile-Ile-Gly-Gly, data not shown). We next screened the
recombinant proteins against a small panel of peptide thiobenzyl ester
substrates to ascertain whether proteolytic activity was present in
either protein (Fig. 2). The wild type
grH demonstrated strong cleavage of Suc-Phe-Leu-Phe-SBzl, less
efficient but reproducible cleavage of Boc-Ala-Ala-Met-SBzl, but no
tryptase (cleavage of Z-Lys-SBzl) or Asp-ase activities. In contrast,
the Ser-to-Ala mutated grH showed no proteolytic activity against any
of the substrates. Together, these data strongly suggested that grH has
chymotrypsin-like activity and that the proteolytic activity purified
from the baculovirus-infected cultures was indeed due to grH.
Kinetic and Inhibitor Studies--
To extend these observations,
we then undertook a kinetic analysis of grH hydrolytic activity against
a more comprehensive panel of synthetic oligopeptide thiobenzyl esters
or aminomethylcoumarin (AMC) substrates (Table
II). The results indicate that grH is a
chymotrypsin-like enzyme which prefers a hydrophobic amino acid residue
(e.g. Phe or Tyr) at the P1 site with Suc-Phe-Leu-Phe-SBzl being the best substrate. GrH can also tolerate an aliphatic residue such as Met, Nva, or Nle at P1 with slower rates. However, there is
very little activity toward substrates which contain Asp, Arg, or Val
at the P1 site. No activity has been detected with the AMC substrates.
The mutant grH does not show any activity toward Suc-Phe-Leu-Phe-SBzl
and Suc-Ala-Ala-Met-SBzl.
Various synthetic inhibitors such as isocoumarins, peptide chloromethyl
ketones, and peptide phosphonates have been previously tested with
other granzymes (23, 24), and the same inhibitors were used to test
recombinant grH (Table III). The most
effective inhibitor was the general serine protease inhibitor DCI which completely inhibited grH at 0.1 mM after 10 min of
pre-incubation. PMSF also showed some inhibition, but at a higher
inhibitor concentration (1 mM). Peptide phosphonates and
peptide chloromethyl ketones which contain the Phe-Leu-Phe sequence
were also moderate inhibitors of grH at 0.1 mM
concentration. Interestingly, the most effective phosphonate inhibitor
was FTC-Aca-Phe-Leu-Phe(P)(OPh)2. This indicates that grH
may have a remote subsite for very hydrophobic structures.
Fluorescinated grH Can Be Internalized into Endosome-like Vesicles
in Jurkat Cells--
It has recently been shown that the binding of
radioiodinated grB to putative target cell surface receptors is
saturable and can be blocked by an excess of unlabeled grB (25).
Furthermore, cell surface binding of grB is followed by
temperature-dependent uptake into Rab4-positive early
endocytic vesicles (26). To determine whether recombinant grH could be
internalized in a manner analogous to grB, we exposed healthy Jurkat
cells to recombinant, enzymatically active grH which had been labeled
with fluorescein (FITC-grH), and examined cellular binding and uptake
of grH using confocal microscopy (Fig.
3). After incubation at 4 °C for 10 min, we observed cell surface binding of FITC-grH that was similar in
appearance to that seen with FITC-grB. We observed punctate cell
surface fluorescence with both enzymes, however the staining seen with
FITC-grB was slightly more coarse than for FITC-grH. Furthermore, when
the cells were warmed to 37 °C, uptake of both grB and grH was seen
in well defined cytoplasmic vesicles highly suggestive of endosomes.
Very similar patterns of staining were also observed with
FITC-transferrin, which is taken up via clathrin-mediated endocytosis
following association with its cognate receptor; however, there was no
uptake of FITC-labeled 20-kDa dextran at either temperature (Ref. 9,
and data not shown). Incubation of Jurkat cells with Ser-to-Ala-mutated
grH resulted in a reduction, but only incomplete inhibition of uptake
(data not shown). As seen previously with grB in the absence of
perforin, the uptake of grH into endosomes was not associated with any
apparent toxicity of the Jurkat cells, which remained indefinitely
viable under the conditions of study (data not shown). Overall, our
data suggested that grB and grH are both capable of uptake into target
cells by similar mechanisms, but that neither granzyme is capable of
inflicting cell death when applied to the surface of cells without
other pro-apoptotic mediators such as perforin.
Although granzymes were first isolated many years ago (27), the
inability to express them in large quantities and to assess the effects
of site-specific mutations has hindered the study of their complex and
varied biological functions. Granzymes are conventionally purified from
cultures of cytolytic lymphocytes or NK leukemia cell lines that
co-express many proteolytic enzymes with different specificities (2, 3,
5, 18). Given the close physico-chemical similarities between various
granzymes (especially grB and grH), it has been difficult to ensure
that apparently pure granzyme preparations are not contaminated with minuscule quantities of other proteases. Thus, it has been argued that
small amounts of rat RNK-chymase-1 co-purified with perforin or grB
might be responsible for amplifying the pro-apoptotic synergy observed
with these two molecules, perhaps by modifying the structure of
perforin (14). The availability of large quantities of recombinant granzymes should enable such issues to be resolved and, additionally, should provide sufficient purified enzyme to attempt formal structural analysis by crystallographic or other means. Previous studies based on
x-ray crystallographic studies have demonstrated that Gly226 of chymotrypsin A is important for conferring its
chymase activity, and this observation has been used to design grM
mutants that lose their ability to cleave at Met but acquire
chymotrypsin-like activity (28). As with the equivalent residue in
granzymes, Gly226 is situated at the base of the predicted
substrate pocket of chymotrypsin A, and the small size of Gly is
postulated to permit bulky hydrophobic side chains to be accommodated
and cleaved. In an analogous manner, it has been predicted using
computer modeling that Arg208 occupies the corresponding
position in grB (29). This residue has indeed recently been shown to be
important for conferring the substrate specificity of grB, in that it
is capable of electrostatically attracting the negatively charged
acidic side chains of Asp or Glu (29). It is interesting to note that
position 208 of the structurally related grH is Gly, in keeping with
the observed chymotrypsin-like activity of this granzyme, as described
in the present study.
Like many chymases, grH is capable of cleaving polypeptides at a
variety of different P1 amino acids. While our study demonstrated a
clear preference for Phe or Tyr but not Trp, a number of smaller, unbranched hydrophobic or even polar residues were cleaved with some
efficiency. Interestingly, the rat RNK-chymase-1 has specificity for
Phe and Trp, but does not cleave efficiently after Tyr residues (14).
If grH and RNK-chymase-1 have analogous functions and thus cleave
common substrates in their respective species, this variation in
cleavage preference may be useful in delineating candidate cleavage
sites of substrate proteins. By contrast with these two chymases, grB
and grA cleave target proteins almost exclusively at acidic (Asp, Glu)
or basic (Lys, Arg) residues, respectively (2, 3). Another possibility
consequence of the broad spectrum of cleavage for grH and RNK-chymase-1
is that their physiological substrates might possibly be targeted at a number of different sites, but this prediction awaits verification when
cellular substrates of these two granzymes are identified and the
cleavage sites defined.
Our studies have also demonstrated for the first time that like grB (9,
30) and grA (31), grH is potentially capable of uptake into the target
cell cytoplasm in vesicles highly reminiscent of those containing grB.
This finding raises many issues related to the intracellular
trafficking and ultimately the functions of granzymes. First, what is
the nature of cell surface granzyme receptors, and do specific
receptors exist for granzymes of different proteolytic specificity?
Second, is there a requirement for proteolytic activity of a granzyme
for its cellular uptake and trafficking between intracellular
compartments? Third, can different granzymes be taken up into the same
endosome? And finally, what are the physiological function of granzymes
other than grB inside target cells? We have recently shown that the
cytolytic granules of CTL/NK cells contain multiple different pathways
for achieving target cell apoptosis. Some of these pathways rely on the
Asp-ase activity of grB and/or the activation of caspases; however,
cell death can still proceed with remarkable efficiency independently
of the Asp-ase activity of grB, as indicated both by our own work with
purified pro-apoptotic molecules and findings in vivo with grB-deficient mice (32). Intriguingly, apoptosis occurring
independently of grB can occur through a non-nuclear pathway,
suggesting that cytoplasmic or cell membrane substrates of alternative
granzymes might be instrumental in mediating this form of cell death
(33, 34). The ability to produce large amounts of granzyme of various specificity by recombinant means should facilitate the study of this
and related questions.
*
The work outlined in this manuscript has been supported by a
project grant and a Senior Research Fellowship (to J. A. T.) from the
National Health and Medical Research Council of Australia, an
Australian Postgraduate Award (to K. M. E.), and Grant GM 54401 from
the National Institutes of Health (to J. C. P.).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: The John Connell
Laboratory, The Austin Research Institute, Studley Road, Heidelberg, 3084, Australia. Tel.: +61-3-9287-0653; Fax: +61-3-9287-0600; E-mail:
j.trapani@ari.unimelb.edu.au.
The abbreviations used are:
CTL, cytotoxic T
lymphocyte;
Aca, 6-aminocaproyl;
AMC, 7-amino-4-methylcoumarin;
Boc, t-butyloxycarbonyl;
DCI, 3,4-dichloroisocoumarin;
DTNB, 5,5'-dithiobis(2-nitrobenzoic acid);
FITC-grB, fluoresceinated granzyme
B;
FTC, 5-fluoresceinyl(thiocarbamoyl);
GST, glutathione
S-transferase;
IC, isocoumarin;
NK, natural killer;
Nle, norleucine;
Nva, norvaline;
PMSF, phenylmethanesulfonyl fluoride;
SBzl, thiobenzyl ester;
Suc, succinyl;
Z, benzyloxycarbonyl;
PAGE, polyacrylamide gel electrophoresis.
The Human Cytotoxic T Cell Granule Serine Protease Granzyme H
Has Chymotrypsin-like (Chymase) Activity and Is Taken Up into
Cytoplasmic Vesicles Reminiscent of Granzyme B-containing
Endosomes*
,¶
The John Connell Laboratory, The Austin
Research Institute, Studley Road, Heidelberg, 3084, Australia and
the § School of Chemistry and Biochemistry, Georgia
Institute of Technology, Atlanta, Georgia 30332-0400
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C. The yield was
calculated spectrophotometrically at 280 nm, using the theoretical
extinction coefficient of 1.0, according to Gill and von Hippel (20),
and confirmed by SDS-PAGE and silver staining in comparison with known quantities of protein standards.
405 = 13,260 M
1
cm1) on a Molecular Devices microplate reader after 10 µl of enzyme stock (grH, 285 µg/ml; mutant, 290 µg/ml) was added
to a well containing 0.2 ml buffer, 10 µl of DTNB (5 mM),
and 10 µl of substrate (2.5-5.6 mM). The hydrolysis of
AMC substrates was monitored by fluorescence change (
ex = 360 nm, em = 465 nm) at 23 °C using a Tecan
Spectrofluor microplate reader.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
[in a new window]
Fig. 1.
A, expression of 32-kDa recombinant grH
in Sf21 culture supernatants. Western blot analysis of culture
supernatants of Sf21 cells 3 days after infection with
baculovirus constructs expressing Ser-to-Ala mutated ("inactive")
grH or wild type enzyme ("active"), probed with anti-grB polyclonal
antiserum. The supernatants from three independent clones derived with
each construct are shown for comparison. Immunostaining of native
32-kDa grB (and possibly grH) in the lysate of the human NK leukemia
cell line YT is shown for comparison. Culture medium from Sf21
cells expressing an irrelevant baculovirus construct produced no signal
in similar analysis (data not shown). B, purification of
inactive (mutated) and active (wild type) grH by Ni-affinity
chromatography. Silver-stained SDS-PAGE of column fractions eluted with
successive 0.5-ml aliquots of imidazole at the concentrations
indicated.
Yields of recombinant grH from a typical purification
View larger version (30K):
[in a new window]
Fig. 2.
. Wild type grH has chymase and weaker Met-ase
activity. Cleavage of oligopeptide thiobenzyl ester substrates by
the indicated concentrations of wild type grH, mutated grH, or purified
native grB at 37 °C for 15 min (see "Materials and Methods").
The values shown for each substrate represent the mean ± standard
error of triplicate assays, and the experiment shown was representative
of three similar experiments. Each substrate was also cleaved with high
efficiency by YT cell lysate (~100 µg/ml), as shown by the optical
density values in each quadrant.
Enzymatic hydrolysis rates of peptide thioester and AMC substrates by
grH
Inhibition of grH by various inhibitors
View larger version (84K):
[in a new window]
Fig. 3.
Uptake of grH into Jurkat cells mimics the
endocytic uptake of grB. Confocal images of small clusters of
Jurkat cells incubated for 10 min with FITC-grB or FITC-grH at either
4 °C or 37 °C. Cells incubated with FITC-labeled 20-kDa dextran
showed no membrane binding or intracellular uptake of fluorescent label
(data not shown). The experiment shown is representative of four
similar experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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