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From the
Human granzyme A is one of the serine proteinases present in the
granules of cytotoxic T lymphocytes and natural killer cells. Granzymes
are synthesized as inactive proenzymes with an amino-terminal
prodipeptide, which is processed during transport of granzymes to the
cytotoxic granules, where they are stored as active proteinases. In
this study, we explored the possibility of producing recombinant
granzymes. Recombinant human granzyme A zymogen was expressed in
several eukaryotic cell lines (HepG2, Jurkat, and COS-1) after
infection with a recombinant vaccinia virus containing full-length
granzyme A cDNA. Immunoblot analysis of cell lysates showed that all
infected cells produced a disulfide-linked homodimer of identical
molecular weight as natural granzyme A. Infected HepG2 cells produced
the largest amount of this protease (approximately 160 times more than
lymphokine activated killer (LAK) cells). The recombinant protein only
had high mannose type oligosaccharides as did the natural protein.
Although infected HepG2 and COS cells contained high granzyme A antigen
levels, lysates from these cells did not show any granzyme A
proteolytic activity. However, the inactive proenzyme could be
converted into active granzyme A by incubation with the thiol
proteinase cathepsin C (dipeptidyl peptidase I). This study is the
first to demonstrate expression of an active recombinant human
cytotoxic lymphocyte proteinase and conversion of inactive progranzyme
A into an active enzyme by cathepsin C. We suggest that a similar
approach can be used for the production of other granzymes and related
proteinases.
Activated cytotoxic T lymphocytes and natural killer cells
contain specialized cytoplasmic granules, which are able to lyse
susceptible targets(1, 2) . These so called
``cytotoxic granules'' among others contain the pore-forming
protein perforin (3) and a family of highly homologous serine
proteinases, termed granzymes(4) . Seven different granzymes
have been identified in the mouse (granzymes A-G) (4, 5) and four in humans: granzyme A, granzyme B,
granzyme H, and granzyme 3 (6, 7, 8, 9, 10, 11, 12, 13) .
Experiments with purified proteins as well as with knockout mice have
indicated a direct involvement of granzymes A and B in target cell DNA
fragmentation and
apoptosis(14, 15, 16, 17) . Human
and mouse granzymes all contain the catalytic triad consisting of
histidine, aspartic acid, and serine, typical for serine proteinases (4, 5) . Furthermore, six cysteine residues involved
in intrachain disulfide bond formation are conserved among the
granzymes. The structure of granzyme A is unique among the granzymes in
that it contains an additional disulfide bond and forms
disulfide-linked homodimers via a free cysteine residue at position
76(6, 9) . The nucleotide sequence of human granzyme A
predicts a proteinase of 262 amino acids, a 26-amino acid hydrophobic
prepeptide and a short propeptide consisting of a glutamic acid and a
lysine residue. Amino-terminal sequence analysis of granzyme A purified
from cytotoxic granules revealed that it is stored as a fully
processed, active, disulfide-linked, 50-kDa homodimer, i.e. without pre- and propeptide(4, 6, 9) .
Both catalytic centers of the homodimer are active (18) and
preferably cleave synthetic substrates after lysine or
arginine(6, 19) . Granzymes are structurally
related to granular proteinases from myeloid leucocytes such as
cathepsin G, elastase, and mast cell proteinases. Also, these
proteinases are synthesized as inactive precursor molecules and stored
in the granules as active proteinases(4, 6) .
Furthermore, they also have a propeptide consisting of two charged
amino acid residues(20, 21, 22) . In
addition, there is indirect evidence that the thiol proteinase
cathepsin C (also known as dipeptidyl peptidase I) is able to process
these propeptides, as inhibition of this enzyme impairs the generation
of active cathepsin G, elastase, or granzyme A(21) . We have
expressed granzymes A and B in prokaryotic systems(23) , but
recombinant proteins obtained in this way were inactive because of
incomplete folding and aggregation. Here we describe the expression of
recombinant human granzyme A (rGA) (
Figure 1:
A schematic
representation of the vaccinia recombination vector p11k-ATA-18
containing full-length granzyme A cDNA. L-TK and R-TK represent the left and right thymidine kinase locus. The 11k late
promotor is indicated. The arrows on top indicate the
cleavage sites for the signal and pro- (EK)
peptide.
Granzyme A
recombinant virus was prepared by homologous recombination of the
granzyme A/p11k-ATA-18 plasmid with the temperature-sensitive vaccinia
virus mutant ts7. Plaques were selected and purified as
described(26) . Briefly, subconfluent plates of human 143 tk
As a control, a recombinant vaccinia virus containing an
insert coding for the serine proteinase domain of human factor-XII
(rFXII.lpc) was used(27) . Alternatively, the virus was omitted
during the incubation.
Figure 2:
Immunoblot analysis (using a monoclonal
antibody directed against granzyme A, GrA-8) of the granzyme A content
of cell lysates (l) and supernatant (m) of Jurkat (lanes 3-8) and HepG2 cells (lanes 9-14)
infected either with granzyme A recombinant virus (lanes 3-6 and lanes 9-12) or with a control virus (FXII, lanes 7-8 and lanes 13-14). As a positive
control, natural granzyme A-containing LAK cells are analyzed in lanes 1 and 2. The even lanes contain
reduced samples, and the odd lanes contain nonreduced samples.
The following number of cells or cell equivalents (in case of medium)
per lane were analyzed: lanes 1 and 2, 1
Each granzyme A
monomer has one glycosylation site to which an N-linked high
mannose oligosaccharide is bound(9, 29) . No
difference in Endo H susceptibility between LAK cell-derived granzyme A (Fig. 3, lanes 1 and 2) and rGA dimer from
Jurkat or HepG2 was found (lanes 5 and 8,
respectively). Additionally, digestion of rGA with PNGase F, which
cleaves high mannose as well as complex-type sugar chains, yielded a
similar shift in M
Figure 3:
Immunoblot showing PNGase F (PF)
and Endo H (EH) digestion of granzyme A in LAK cells (lanes 1 and 2), infected HepG2 (lanes
3-5) and Jurkat (lanes 6-8) cells. Control
samples(-) were treated identically except that no enzyme was
added.
Compared with the number of LAK cells (Fig. 2, lanes 1 and 2), 53 times fewer HepG2 cells (lanes
9 and 10) and 10 times fewer Jurkat cells (lanes 3 and 4) were analyzed, whereas the amount of HepG2 cell
supernatant tested (lanes 11 and 12) corresponded to
10 times fewer cells. However, in spite of testing a lower number of
infected cells, the intensity of protein bands rGA (compare, for
example, lanes 2 and 10, Fig. 2), was equal to
or higher than that observed with LAK cells, suggesting the latter
produced less granzyme A than the infected cells. This was further
assessed semiquantitatively using an immunoblot; per given amount of
cells, infected Jurkat cells produced approximately 20 times, COS-1 40
times, and HepG2 cells even 160 times more granzyme A antigen (48 h
postinfection) than did LAK cells.
Figure 4:
Binding of natural granzyme A of LAK cells (lanes 2 and 3) and rGA of infected HepG2 (lanes
5 and 6) and Jurkat cells (lanes 8 and 9) to benzamidin-Sepharose in the presence (+) or
absence(-) of PMSF. 80
Figure 5:
A, activation of rGA from infected HepG2
cells by cathepsin C. Lysates of LAK cells or HepG2 cells were
incubated with cathepsin C in the presence of cysteine, after which
binding of granzyme A to benzamidin-Sepharose was analyzed on
immunoblot (lanes 3 and 6, respectively). HepG2 cell
lysate incubated with buffer alone and absorbed to benzamidin-Sepharose
is shown as control (lane 7). In addition, cell lysates of LAK
cells (lane 1) and HepG2 cells (lane 4), not
incubated with cathepsin C, as well as their benzamidin-bound fraction (lanes 2 and 5, respectively) were analyzed. All
lanes contain lysates or absorbed fractions equivalent to 200
The effect of cathepsin C on the
proteolytic activity of rGA in HepG2 cell lysates was also assessed (Fig. 5B). This proteolytic activity increased during
incubation with cathepsin C, whereas no increase of BLT activity by rGA
was detected when cathepsin C was omitted. It is to be noted that
cathepsin C itself, at the concentrations used, did not convert the BLT
substrate (not shown). Thus, together these results demonstrated that
cathepsin C was able to convert recombinant granzyme A in HepG2 cell
lysates into a proteolytically active enzyme. Here we report the expression of recombinant human granzyme A
in mammalian cells by a granzyme A recombinant vaccinia virus and the
ability of cathepsin C to convert rGA zymogen into an active
proteinase. Using immunoblotting, we estimated that infected HepG2
cells produced about 160 times more granzyme A than LAK cells. These
levels exceeded that produced by transient expression in COS cells
using a mammalian expression vector (not shown). This is in concordance
with previous studies comparing conventional transient expression
systems with expression by vaccinia virus(30) . Different
cell lines were infected by recombinant vaccinia virus harboring cDNA
coding for full-length granzyme A including the signal and propeptide.
Infected cell lines produced a disulfide-linked homodimer with the same
molecular weight as natural granzyme A. In vitro translation
of granzyme A mRNA, using rabbit reticulocytes and dog microsomes in
the presence of oxidized glutathione, produced a granzyme A dimer
unable to bind to benzamidin-Sepharose. ( Granzymes are synthesized as inactive precursor molecules
with a short prodipeptide consisting of Glu-Lys in the case of human
granzyme A and of Gly-Glu in human granzymes B and H and mouse granzyme
B. Mast cell and neutrophil serine proteinases contain similar, short
acidic propeptides. Formation of proteolytically active human elastase
and cathepsin G involves a dual proteolytic processing pathway. First,
the signal peptide is cleaved off, generating an inactive zymogen;
second, the amino-terminal dipeptide and a carboxyl-terminal extension
are removed, thereby generating active enzyme(20) . Although
rGA produced by vaccinia-infected cells consisted of a homodimer with
similar molecular weight as natural granzyme A, it had no proteolytic
activity, except for rGA produced by Jurkat cells. Similar observations
have been made for COS cells transfected with full-length cDNA coding
for human leukocyte elastase or murine granzyme B
cDNA(22, 33) , whereas COS cells transfected with a
mutant granzyme B cDNA lacking the prodipeptide did produce active
granzyme B(22) . Together these data suggest that HepG2 and
monkey COS cells, in contrast to Jurkat cells, lack the ability to
process the prodipeptide of granzymes and related proteinases
correctly. It has been suggested that the lysosomal cysteine
proteinase dipeptidyl peptidase I, previously termed cathepsin C, is
the putative enzyme involved in the processing of the amino-terminal
propeptides of granzymes and myeloid associated serine
proteinases(21) . In agreement with that, high levels of
cathepsin C occur in the spleen and other lymphoid or myeloid
cells(34, 35) . Thus, the inability of COS and HepG2
cells infected with recombinant vaccinia virus, coding for the
full-length granzyme A cDNA, to generate active rGA may have been due
to low levels of cathepsin C in these cells. In contrast, Jurkat cells,
a human T-helper leukemia cell line, constitutively express granzyme A (9) and thus presumably contain cathepsin C. Part of the rGA
produced by infected Jurkat cells indeed appeared to be active,
although this activity was only 20% compared with natural granzyme A.
Overproduction of recombinant protein, together with suppression of
host protein synthesis by the viral infection, may have disturbed
complete processing of recombinant progranzyme A by Jurkat cells. Activation of serine proteases results from the ability of the
This study is the first to
demonstrate expression of an active recombinant human cytotoxic
lymphocyte proteinase. By expressing rGA as an inactive zymogen,
proteolytic damage to the expression system was minimized, thereby
allowing high expression levels. Furthermore, the cysteine proteinase
cathepsin C was shown to be able to convert rGA zymogen into an active
enzyme, implying that cathepsin C may be involved in the processing of
natural granzymes. A similar strategy may be feasible for the
expression of other granzymes or related proteinases.
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9281-9286
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)as a zymogen by HepG2
cells infected with a granzyme A recombinant vaccinia virus. Granzyme A
zymogen could be converted into an active enzyme by cathepsin C,
providing further evidence for the involvement of cathepsin C in the
processing of granzyme A.
Reagents
Monoclonal antibody GrA-8, directed
against human granzyme A, was produced as described
previously(23) . Horseradish peroxidase-conjugated goat
anti-mouse immunoglobulins were obtained from the Department of Immune
Reagents of our institute (CLB, Amsterdam), the chemiluminescence
detection kit from Amersham International (Buckinghamshire, United
Kingdom), and bovine cathepsin C, endoglycosidase H (Endo H) and
peptide-N-glycosidase F (PNGase F) from Boehringer Mannheim.
Benzamidin-Sepharose was purchased from Pharmacia Fine Chemicals
(Uppsala, Sweden), N
-benzyloxycarbonyl-L-lysine
thiobenzyl ester (BLT) from Calbiochem, and the chromogenic substrate
S2288 (D-Ile-Pro-Arg-pNA) from Kabi Diagnostica (Stockholm,
Sweden). Nonidet P-40 and phenylmethylsulfonyl fluoride (PMSF) were
obtained from Sigma.Cell Culture
Human 143 tk
fibroblasts and RK-13 rabbit kidney cells were maintained in
Eagle's medium supplemented with 10% (v/v) fetal calf serum,
streptamycin, penicillin, and nonessential amino acids. SV
40-transformed COS-1 monkey cells, the human T-helper cell line Jurkat,
and the human liver cell line HepG2 were grown in Iscove's
modified Dulbecco's medium supplemented with 10% (v/v)
heat-inactivated fetal calf serum, streptamycin, penicillin, and
-mercaptoethanol (medium for Jurkat cells also contained 20
units/ml interleukin-2; Chiron, Emeryville, CA). Lymphokine-activated
killer (LAK) cells were prepared by culturing human peripheral blood
mononuclear cells (obtained from healthy donors by Percoll density
gradient centrifugation) at a concentration of 0.5 
10
cells/ml for 7 days with 1,000 units/ml interleukin-2 in the same
medium as for Jurkat cells. LAK cell lysate (20 10 cells/ml) was prepared as described below for vacciniainfected
cells.Construction of Recombinant Vaccinia Virus for the
Expression of Human Granzyme A
To obtain full-length granzyme A
cDNA, including the nucleotides coding for the pre- and propeptide,
specific primers containing the appropriate restriction sites were
prepared based on the published granzyme A cDNA sequence(9) .
First strand cDNA was prepared from mRNA of LAK cells as described (23) and amplified with the polymerase chain reaction using the
granzyme A primers. The amplified cDNA fragment was isolated, digested
with the appropriate restriction enzymes, and ligated into the SacI SphI sites of the vaccinia virus
recombinant vector p11k-ATA-18 (24) (Fig. 1). The
authenticity of the cloned cDNA was confirmed by nucleotide sequence
analysis (Sequenase kit; U.S. Biochemical Corp.). Isolation of plasmid
DNA, conditions for digestion by restriction enzymes, and agarose gel
electrophoresis were as described(25) .
fibroblasts were infected for 1 h with
vaccinia virus ts7 (0.1 plaque-forming unit/cell). Subsequently, cells
were incubated with fresh medium for 2 h at the permissive temperature
of 33 °C, transfected with a calcium phosphate co-precipitate of
wild type vaccinia virus DNA (100 ng/10
cells) and an
equivalent amount of recombinant plasmid DNA, and incubated for 2 h at
the nonpermissive temperature, 39.5 °C. Medium was then removed,
and cells were rinsed and incubated for 48 h at 39.5 °C. Infected
cells and culture medium were collected and freeze-thawed once.
Dilutions of this material, containing recombinant virus, were used to
select for tk viral plaques by infecting
human 143 tk fibroblast cultures incubated
in the presence of bromodeoxyuridine (100 µg/ml). Single plaques
were purified and amplified in RK-13 cells and stored at -20
°C. This viral stock was titrated on RK-13 cells to determine the
number of plaque-forming units/ml of virus stock.Granzyme A Expression in Different Cell Lines
Cell
lines were infected with granzyme A recombinant vaccinia virus by
incubating subconfluent monolayers of HepG2 or COS-1 cells in
10-cm
dishes with 10 plaque-forming units of recombinant
virus/cell. Jurkat cells, suspended at 10 10 cells/ml, were incubated with 500 plaque-forming units/cell for
60 min at 37 °C, diluted 10-fold with fresh medium, and then
further incubated for 24 or 48 h at 37 °C. Thereafter, cells and
supernatant were harvested. The supernatant was centrifuged at 3,000
rpm to remove cellular debris, incubated with 1% (w/v, final
concentration) Nonidet P-40 to inactivate viral particles, and stored
at -70 °C for further analysis. Cells were washed with
phosphate-buffered saline (PBS), resuspended at a concentration of 2
10 cells/ml in ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% (w/v) Nonidet P-40),
gently mixed, and left for 30 min in melting ice. The mixture was then
centrifuged for 10 min at 200 g (4 °C) to remove
the nuclei and the supernatant was stored at -70 °C until
used.Enzyme Assays
Granzyme A proteolytic activity was
measured essentially as described(28) . Briefly, 20 µl of
cell lysate was added to 100 µl of 0.1 M Tris-HCl, pH 8.0,
0.5% (v/v) Nonidet P-40, 0.3 mM BLT, and 0.3 mM dithiobis(2-nitrobenzoic acid) (Aldrich-Chemie, Steinheim,
Germany). The absorbance at 414 nm was read over 1 h at 37 °C on a
microplate reader. The amount of granzyme A activity was expressed as
the increase of absorption at 414 nm/min.Glycosidase Treatment
Total cell lysates (with a
final concentration of 0.25% (v/v) Nonidet P-40) were first denatured
in 0.5% (w/v) SDS at 95 °C for 5 min, after which the SDS was
quenched by addition of a 2-fold excess of Nonidet P-40. Then PNGase F
at a final concentration of 3 milliunits/ml was added for 16 h at
37° C in the presence of 2 mM PMSF. Similarly, samples
were treated with endo H by incubation for 16 h at 37 °C in 9
mM NaAc, pH 5.5, in the presence of 2 mM PMSF and 3
milliunits of the enzyme/ml. All samples were centrifuged at 13,000 rpm
for 2 min, prior to analysis by immunoblotting.Activation of rGA Zymogen by Cathepsin C
LAK cell
lysate or supernatant from HepG2 cells infected (for 48 h) with
granzyme A or FXII.lpc recombinant viruses was dialyzed against 50
mM NaAc, pH 5.0, 30 mM NaCl, 1 mM EDTA, 10
mM cysteine (Pierce) at 4 °C. Samples were then incubated
for varying time intervals at 37 °C with or without 0.02 units of
purified bovine cathepsin C. Samples were neutralized and stored on ice
until analyzed for granzyme A activity. This activity was analyzed
either by incubating samples with benzamidin-Sepharose, whereafter
bound fraction (i.e. active granzyme A) was analyzed on
immunoblot (see below), or by determining the conversion of BLT
substrate. To prevent interference of cysteine in the chromogenic
assay, samples were dialyzed against PBS at 4 °C prior to analysis. Affinity Purification of Granzyme A Species Using
Benzamidin-Sepharose
Cell lysates or supernatant was diluted in
lysis buffer to a final volume of 250-500 µl and incubated
with 10 µl of benzamidin-Sepharose for 4 h at room temperature on a
head-over-head rotator. The Sepharose beads were then washed four times
with 1 ml of lysis buffer. The supernatant of the last washing step was
carefully removed, after which SDS-sample buffer was added to the
Sepharose beads. The mixtures were incubated for 5 min at 100 °C
and centrifuged for 3 min at 13,000 g. The supernatant
was electrophoresed on SDS-polyacrylamide (12.5%, w/v) gels and
subsequently analyzed by immunoblot (see below).Immunoblotting
Cell lysates or
benzamidin-Sepharose precipitates were separated on 12.5% (w/v)
polyacrylamide gels. Proteins were then transferred onto nitrocellulose
sheets (Schleicher and Schuell), which then were incubated for 30 min
with blocking buffer, i.e. PBS containing 5% (w/v) nonfat dry
milk (Protifar; Nutricia, Zoetermeer, the Netherlands) and 0.1% (w/v)
Tween 20. The sheets were then incubated with monoclonal antibody GrA-8
(at 2.5 µg/ml in the same buffer), for 14 h at room temperature.
Sheets were washed by repeated (3 times) incubation for 10 min with
PBS, 0.1% (w/v) Tween 20, and then probed with horseradish
peroxidase-conjugated goat anti-mouse immunoglobulins diluted in
blocking buffer for 2 h. After a wash with PBS, 0.1% (w/v) Tween, and
one with PBS alone, sheets were developed for 2 min in chemiluminescent
detection reagent (Amersham International) and exposed for 15-120
s to Kodak XS1 films (Kodak, Rochester, NY).
Construction of a Granzyme A Recombinant Vaccinia
Virus
A full-length granzyme A cDNA was cloned from activated T
lymphocytes as described under ``Materials and Methods.'' The
nucleotide sequence of the clone obtained was identical to that
published by Gershenfeld et al.(9) . The cDNA was
inserted into the vaccinia virus recombination vector p11k-ATA-18,
under the control of the 11k late promotor (24) (Fig. 1). It encoded the complete protein including
the signal peptide and the prodipeptide (Fig. 1). The cDNA was
intergrated into the viral DNA of the wild type virus by recombination
as described elsewhere(24) . The promotor used enables the
synthesis of a large amount of foreign polypeptides in the late phase
of the viral infection, initiating translation at their authentic start
codon.Characterization of rGA Produced by Different Cell
Lines
Jurkat, HepG2, and COS-1 cells were infected with rGA
vaccinia virus. Twenty-four and 48 h after infection all cell lines
produced granzyme A protein. Cell lysates as well as culture
supernatants from Jurkat or HepG2 cells obtained 48 h after infection
appeared to contain rGA only when infected with granzyme A recombinant
virus (Fig. 2, lanes 3-6 and lanes
9-12, respectively), but not after infection with a control
virus (Fig. 2, lanes 7-8 and lanes
13-14, respectively). RecGran A consisted of a
disulfide-linked homodimer with a relative mobility (M
) identical to the natural protein (LAK cells; Fig. 2, lanes 1 and 2).
10; lanes 3-8 and lanes 11 and 12, 0.16 10; lanes 9 and 10 and lanes 13 and 14, 0.03
10.
as treatment with Endo H,
indicating that glycosylation of rGA was identical with that of the
natural protein, i.e. of the high mannose type. Deglycosylated
rGA protein from Jurkat and HepG2 cells migrated with the same M compared with deglycosylated granzyme A from LAK
cells, indicating that the molecular weight of the protein backbone of
the recombinant species was similar to that of natural granzyme A.
The Amount of rGA Produced by HepG2 Is Considerably
Higher than That Produced by LAK Cells
In general, infected
cells produced a maximal amount of rGA antigen 48 h postinfection.
During infection an increase of cell lysis and a concomitant release of
rGA into the supernatant was observed (Fig. 2, lanes 11 and 12). The extent of cell lysis and subsequent granzyme
A release into the supernatant varied during different infection
experiments.Recombinant Granzyme A Shows No Proteolytic
Activity
Enzymatic activity of the recombinant protein was
determined in two ways; first, cell lysates were tested for hydrolysis
of the chromogenic substrate BLT, and, second, the affinity of
recombinant proteins for benzamidin-Sepharose was determined, as
benzamidin only binds to proteolytically active granzyme A. In these
experiments LAK, HepG2, Jurkat, and COS-1 cell lysates were adjusted to
contain approximately equal amount of granzyme A antigen as assessed by
immunoblotting. Lysates from COS-1 or HepG2 cells showed hardly any BLT
hydrolysis (the activity corresponded to less than 0.5% of that of LAK
cells; Table 1). In agreement herewith, rGA from HepG2 cells did
not bind to benzamidin-Sepharose (Fig. 4, lane 5),
whereas natural granzyme A from LAK cells did (Fig. 4, lane
2). In contrast, lysates of infected Jurkat cells had significant
BLT activity, corresponding to about 20% of that present in LAK cell
lysates (Table 1). No BLT activity was observed in lysates from
Jurkat cells infected with FXII.lpc recombinant virus, consistent with
the observed lack of granzyme A antigen (Fig. 2, lanes 7 and 8). The BLT activity observed with infected Jurkat
cells was due to the presence of proteolytically active rGA, since part
of the latter bound to benzamidin-Sepharose (Fig. 4, lanes 7 and 8). Preincubation of granzyme A from LAK cells or
infected Jurkat cells with PMSF, an inhibitor of serine proteinases,
prevented binding to benzamidin-Sepharose, demonstrating the
specificity of this binding (Fig. 4, lanes 3 and 9, respectively).
10
LAK, 0.5
10 HepG2, and 8 10 Jurkat cells were
incubated for 4 h with bezamidin-Sepharose, and the granzyme A bound
was analyzed on immunoblot as described under ``Materials and
Methods.'' In addition, for each cell type a complete lysate (cl) of equal cell numbers was tested (lanes 1, 4, and 7).
Recombinant Granzyme A Is Converted into a Proteolytic
Active Species by Cathepsin C
Although rGA from HepG2 displayed
similar biochemical features (i.e. M,
dimerization, and high-mannose type glycosylation) as natural granzyme
A from cytotoxic cells, hardly any proteolytic activity toward BLT
substrate was observed. Presumably, the inability of HepG2 cells to
produce proteolytically active granzyme A was due to inappropriate
processing of the activation dipeptide. Therefore, rGA-containing HepG2
cell lysates were incubated with the lysosomal cysteine proteinase
cathepsin C and assessed for granzyme activity (Fig. 5). Upon
incubation with cathepsin C, rGA in HepG2 cell lysates appeared to bind
to benzamidin-Sepharose (Fig. 5A, lane 6),
whereas this was not observed upon incubation of rGA with buffer alone (lane 7). No difference in M was observed
prior to and after cathepsin C treatment (lanes 4 and 6, respectively), indicating only a minor modification of rGA
occurred after cathepsin C treatment. As expected, natural granzyme A
from LAK cell lysates was resistant to cathepsin C treatment, as
incubation with cathepsin C did not alter reactivity toward
benzamidin-Sepharose (lanes 2 and 3). Apparently,
after processing of the prodipeptide, cathepsin C was not able to
further modify active granzyme A.
10
LAK or 5 10 HepG2 cells. B,
time course of activation of rGA from infected HepG2 cells by cathepsin
C. The amount of BLT hydrolysis is expressed as LAK cell equivalents.
HepG2 cell lysate is incubated with cathepsin C and cysteine (
),
with cysteine alone (
), or without cathepsin C and cysteine
(
), as described under ``Materials and
Methods.''
)Apparently,
dimerization can occur before formation of active granzyme A.
Furthermore, these in vitro translation experiments showed
that dimerization of granzyme A likely takes place in the rough
endoplasmic reticulum, i.e. before the proteinase is
activated. Experiments with PNGase F and Endo H showed that rGA only
contained high mannose-type oligosaccharides, similar to natural
granzyme A. Binding of the mannose 6-phosphorylated sugar to the
mannose 6-phosphate receptor leads to selective transport of granzyme A
to the cytotoxic granules(31, 32) . The majority of
the rGA produced by HepG2 or COS cells was retained in the infected
cells. Immunofluorescence showed a granular staining pattern (not
shown) comparable with that in LAK cells(23) , suggesting
recombinant protein was targeted to lysosomes. Alternatively,
overproduction of recombinant protein, combined with the virus
infection, may have destroyed the cellular architecture, leading to an
accumulation of the protein in the endoplasmic reticulum or the Golgi
apparatus.
-amino group of the first isoleucin of the mature enzyme (Fig. 1), generated after processing of the propeptide, to form
an ion pair with the aspartic acid of the catalytic pocket. This
interaction enables the formation of a functional catalytic
center(20) . Apparently, after removal of propeptide by
cathepsin C, rGA had proteolytic activity. No decrease in esterolytic
activity or loss in affinity for benzamidin-Sepharose was observed
after prolonged cathepsin C treatment of natural or activated rGA. This
indicates that active granzyme A is refractory to further cathepsin C
treatment. If cathepsin C-mediated N-terminal dipeptide cleavage would
proceed, the protease would lose its stable conformation and,
therefore, its proteolytic activity.
)
-benzyloxycarbonyl-L-lysine
thiobenzyl ester; Endo H, endoglycosidase H; LAK, lymphokine-activated
killer; PBS, phosphate-buffered saline, pH 7.4; PMSF,
phenylmethylsulfonyl fluoride; PNGase F, peptide-N-glycosidase
F.)
We thank Florine van Milligen and Eric Eldering for
critically reading the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 V. R. Sutton, N. J. Waterhouse, K. A. Browne, K. Sedelies, A. Ciccone, D. Anthony, A. Koskinen, A. Mullbacher, and J. A. Trapani Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis J. Cell Biol., February 12, 2007; 176(4): 425 - 433. [Abstract] [Full Text] [PDF]
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 N. Bidere, M. Briet, A. Durrbach, C. Dumont, J. Feldmann, B. Charpentier, G. de Saint-Basile, and A. Senik Selective Inhibition of Dipeptidyl Peptidase I, Not Caspases, Prevents the Partial Processing of Procaspase-3 in CD3-activated Human CD8+ T Lymphocytes J. Biol. Chem., August 23, 2002; 277(35): 32339 - 32347. [Abstract] [Full Text] [PDF]
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 E. H. A. Spaeny-Dekking, A. M. Kamp, C. J. Froelich, and C. E. Hack Extracellular granzyme A, complexed to proteoglycans, is protected against inactivation by protease inhibitors Blood, February 15, 2000; 95(4): 1465 - 1472. [Abstract] [Full Text] [PDF]
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E. Wilharm, M. A. A. Parry, R. Friebel, H. Tschesche, G. Matschiner, C. P. Sommerhoff, and D. E. Jenne Generation of Catalytically Active Granzyme K from Escherichia coli Inclusion Bodies and Identification of Efficient Granzyme K Inhibitors in Human Plasma J. Biol. Chem., September 17, 1999; 274(38): 27331 - 27337. [Abstract] [Full Text] [PDF]
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 C. T. N. Pham and T. J. Ley Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo PNAS, July 20, 1999; 96(15): 8627 - 8632. [Abstract] [Full Text] [PDF]
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D. Garwicz, A. Lindmark, A.-M. Persson, and U. Gullberg On the Role of the Proform-Conformation for Processing and Intracellular Sorting of Human Cathepsin G Blood, August 15, 1998; 92(4): 1415 - 1422. [Abstract] [Full Text] [PDF]
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P. J. Beresford, M. Jaju, R. S. Friedman, M. J. Yoon, and J. Lieberman A Role for Heat Shock Protein 27 in CTL-Mediated Cell Death J. Immunol., July 1, 1998; 161(1): 161 - 167. [Abstract] [Full Text] [PDF]
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C. T. N. Pham, D. A. Thomas, J. D. Mercer, and T. J. Ley Production of Fully Active Recombinant Murine Granzyme B in Yeast J. Biol. Chem., January 16, 1998; 273(3): 1629 - 1633. [Abstract] [Full Text] [PDF]
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P. J. Beresford, C.-M. Kam, J. C. Powers, and J. Lieberman Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them PNAS, August 19, 1997; 94(17): 9285 - 9290. [Abstract] [Full Text] [PDF]
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C. T.N. Pham, R. J. Armstrong, D. B. Zimonjic, N. C. Popescu, D. G. Payan, and T. J. Ley Molecular Cloning, Chromosomal Localization, and Expression of Murine Dipeptidyl Peptidase I J. Biol. Chem., April 18, 1997; 272(16): 10695 - 10703. [Abstract] [Full Text] [PDF]
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P. J. Wolters, C. T. N. Pham, D. J. Muilenburg, T. J. Ley, and G. H. Caughey Dipeptidyl Peptidase I Is Essential for Activation of Mast Cell Chymases, but Not Tryptases, in Mice J. Biol. Chem., May 18, 2001; 276(21): 18551 - 18556. [Abstract] [Full Text] [PDF]
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