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) ()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.
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 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.
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
10
; lanes 3-8 and lanes 11 and 12, 0.16
10
; lanes 9 and 10 and lanes 13 and 14, 0.03
10
.
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 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.
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 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).
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
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.''
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. ()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.
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
-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.
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.