(Received for publication, September 5, 1995; and in revised form, December 4, 1995)
From the
One mechanism used by cytotoxic T cells and natural killer cells to kill target cells involves synergy between the pore-forming protein, perforin, and a serine protease termed granzyme B, both constituents of the cytoplasmic granules of cytolytic lymphocytes. Exposing susceptible cells to perforin and granzyme B results in apoptosis, the morphological consequences of which are most clearly seen in the nucleus. It is conventionally accepted that perforin acts by perforating the target cell membrane; however, the site and mode of action of granzyme B are unknown. We have addressed this issue using Western blotting, proteolytic assays, and confocal laser scanning microscopy to demonstrate that purified human granzyme B can be taken up in large amounts and bound within nuclei. By contrast, perforin and nongranzyme serine proteases did not undergo nuclear uptake. Both unglycosylated human granzyme B (26 kDa) and that bearing high mannose glycosylation (32 kDa) were internalized and bound within nuclei, but forms greater than 32 kDa with complex carbohydrate addition were excluded. The uptake of granzymes was not dependent on net charge, as nuclei absorbed similar quantities of granzyme B at neutral pH and through a range of basic pHs but did not take up other very basic serine proteases such as the mouse mast cell protease 5. Confocal laser scanning microscopy indicated nuclear and nucleolar accumulation of fluoresceinated granzyme B by isolated nuclei. Measurement of the kinetics of nuclear import using an in vitro nuclear transport assay indicated maximal levels of nuclear accumulation of granzyme about 2.5-fold above those in the cytoplasm and nucleolar accumulation a further 3-4-fold higher. Nuclear and nucleolar accumulation were exceedingly rapid, reaching half-maximal levels within 3.3 and 7.5 min, respectively, implying that nuclear accumulation probably occurs prior to transport to the nucleolus. Our observations may provide a mechanism explaining how aspartate-specific cell death proteases access the nuclear substrate poly(ADP-ribose) polymerase, the cleavage of which is an early event in apoptosis.
CTL ()and NK cells utilize at least two mechanisms
for ridding higher organisms of virus-infected, alloreactive, or
transformed cells. One mechanism requires a signal transmitted
following binding of the Fas ligand on the effector cell with its
receptor (Fas/Apo-1/CD95) on the target cell(1, 2) .
Alternatively, targeted cells can be eliminated following their
exposure to the contents of lysosome-like cytoplasmic granules found in
CTL and NK cells (the ``granule exocytosis'' model, (3, 4, 5) ). The end result of both pathways
is apoptotic cell
death(1, 2, 3, 4, 5) . The
profound T cell immunosuppression observed in mice with targeted
mutation of the perforin gene has recently put beyond doubt the pivotal
importance of perforin in immune responses against many intracellular
pathogens and in skin and tumor allograft rejection(6) . By
contrast, natural mutations of the Fas system result in
lymphoproliferative syndromes and autoimmune phenomena(7) .
Perforin is capable of inflicting complement-like pores on target
cell membranes, and in certain instances its sole action can lead to
osmotic lysis(8) . However, purified perforin is unable to
inflict DNA fragmentation and apoptosis, irrespective of the degree of
membrane damage(9, 10) . The involvement of granzymes
in cell death was first suspected on the basis that metabolic
inhibitors of serine proteases could block
cytolysis(11, 12) . More recently it has been shown
that fragmentin-2 (granzyme B) could induce rapid DNA fragmentation,
but only when cells were simultaneously exposed to sublytic quantities
of perforin(13) . These results implied that granzyme B was the
causative agent of apoptosis, but that perforin was indispensable for
granzyme B to gain access to its ligand/substrate. Following exposure
to perforin and granzyme B, some cells undergo a marked up-regulation
of p34 (Cdc2) kinase activity(14) .
Expression of active Cdc2 is normally restricted to the G
/M
phase of the cell cycle, but cells treated with perforin and granzyme B
may show a dislocation of Cdc2 activity from cell cycle
constraints(13, 14) . Proponents of this model suggest
that granzyme B can trigger changes akin to ``mitotic
catastrophe'' observed in yeast cells(15) , therefore
accounting for some of the morphological changes of apoptosis,
particularly dissolution of the nuclear membrane.
Understanding the mechanism of granzyme-induced nucleolysis is dependent on elucidation of natural granzyme substrates. Recently, we demonstrated that granzymes could be detected in the nuclear lysates of human cytolytic lymphocytes and were recoverable from the nuclei in an active form(16) . One crucial issue remaining is whether the granzymes, in particular granzyme B, can be sequestered within nuclei following their release from cytosolic granules, particularly in target cells exposed to granzyme B. In the present study, we have used cell fractionation and confocal laser scanning microscopy (CLSM) to demonstrate that the nuclei of a variety of cells are specifically able to sequester granzyme B, but not non-granzyme serine proteases, and furthermore, that free granzyme B is accumulated within nuclei and nucleoli. Our findings are consistent with the hypothesis that granzymes exert their actions following penetration into the target cell cytoplasm, and introduce the possibility that the nucleus and perhaps nucleolus are physiological sites of granzyme action.
Nuclear transport
was quantified using CLSM as described previously (23, 24, 26) , and image analysis was
performed using the MacIntosh NIH Image 1.49 public domain
software(26) . Passive nuclear transport was routinely
monitored using FITC-labeled dextrans of 20 and 70 kDa (Sigma), while
nuclear localization sequence (NLS)-dependent transport was assessed
using a previously described T-ag--galactosidase fusion protein
(476 kDa) containing the T-ag CcN motif (amino acids 111-135)
including the NLS (amino acids
126-132)(23, 24, 25) .
We have previously demonstrated that nuclear extracts of a variety of human cells and cell lines with NK activity contained active granzymes. By contrast, perforin, which is co-packaged with granzymes in cytoplasmic vesicles, did not localize within the nuclear fraction (16) . The nuclear extracts of cell lines that expressed large amounts of non-granzyme serine proteases, such as the RBL cell line, contained no serine protease activity, suggesting that this phenomenon was peculiar to granzymes. In the present study, the crucial issue was to determine whether granzyme B could be absorbed into the nuclei of cells exposed to granzyme B.
We initially undertook mixing experiments in which nuclei of YAC-1 mouse lymphoma cells were incubated with immunopurified granzyme B (Fig. 1). Following thorough washing, the nuclear proteins were extracted and analyzed by immunoblotting. We estimated that approximately half of the added granzyme B was present in the nuclear lysate, as the signal obtained (lane N) was approximately equal to that of granzyme unbound in the supernatant (lane U). In accord with this estimate, the ability of the recovered granzyme B to cleave the synthetic oligopeptide substrate, Boc-ala-ala-asp-S-benzyl, was approximately equivalent to that of the unbound material (data not shown). As the nuclear pellet occupied <5.0% of the original incubation volume, our data suggested that the granzyme B was being concentrated at least 10-fold over what could be accounted for by simple diffusion. Granzyme B was barely detectable in the wash supernatants on prolonged autoradiography, suggesting that granzyme was tightly bound to the nuclear pellet.
Figure 1: The binding of immunopurified granzyme B to isolated YAC-1 nuclei, demonstrated by Western blot analysis using the anti-granzyme B mAb, 2C5. Unlabeled lane at left, granzyme B added; lane U, granzyme B remaining unbound in the supernatant after pelleting the nuclei; washes 1, 2, and 3, samples of the supernatant after pelleting the nuclei after each wash; lane N, granzyme B localized within the nuclear pellet. For each lane other than those representing the washes, the volumes of sample analyzed have been adjusted to allow a direct evaluation of the amount of granzyme present. By comparison, samples of the wash supernatants 1, 2, and 3 were diluted 10-fold. The markers at left represent the migration of molecular mass standards in kDa.
Figure 2: Binding of granzyme B to various nuclei. A, Western blot analysis, using the anti-granzyme B mAb, 2C5, and demonstrating the binding of granzyme B in unfractionated cytosolic lysate from YT cells (left panel) to isolated YAC-1, HL-60, and RBL nuclei. Unbound (center panel) and bound (right panel) granzyme B species are shown for each nuclear type. The markers at left represent the migration of molecular mass standards in kDa. B, Western blot analysis of supernatant (S) and nuclear (N) fractions from YAC-1 nuclei, following incubation with the YT lysate in A. The antisera used were PB2 (mouse mAb anti-human perforin, panel at left) and anti-BiP (panel at right).
Figure 3: CLSM images of nuclear and nucleolar localized FITC-labeled granzyme B in isolated YAC cell nuclei. A, fluorescent visualization; B, phase contrast view of the same field using the CLSM in transmission mode. Incubation with granzyme B was for 30 min at 4 °C, prior to centrifugation onto glass slides. Results were identical for HL-60 cells (not shown). Fluorescence quantitation by image analysis (average readings of at least 68 nuclei) is shown in C.
These results were confirmed and
elaborated using an in vitro nuclear protein import assay (23) and quantitative CLSM to assess nuclear and nucleolar
import kinetics ( Fig. 4and Fig. 5). Strong nuclear and
particularly nucleolar accumulation of granzyme B was observed (Fig. 4A and Fig. 5A), in contrast to
exclusively nuclear accumulation observed with an IAF-labeled
-galactosidase fusion protein carrying the SV40 T-ag NLS (Fig. 4B and Fig. 5B). As a control for
specificity, another serine protease, FITC-labeled human chymotrypsin,
was found not to accumulate to any extent in either nucleus or
nucleolus (Fig. 4D). It achieved rapid equilibration
between nucleus and cytoplasm, consistent with the fact that its size
(30 kDa) is below the molecular weight cut-off (
45 kDa) for
passive diffusion into and out of the nucleus(27) .
Nucleocytoplasmic equilibration of a FITC-labeled 20-kDa dextran is
shown in Fig. 4C for comparison.
Figure 4:
CLSM images of in vitro nuclear
protein accumulation in mechanically perforated HTC cells. The in
vitro nuclear protein import assay was carried out at room
temperature in the presence of an ATP regenerating system and exogenous
cytosol, as described under ``Materials and
Methods''(23) . A, FITC-labeled granzyme; B, IAF-labeled T-ag--galactosidase-fusion protein; C, FITC-labeled dextran (20 kDa); and D, FITC-labeled
chymotrypsin. Incubation was for 3 (A, C, and D) and 30 min, respectively.
Figure 5:
Time course of nuclear and nucleolar
import of FITC-labeled granzyme B (A) and IAF-labeled
T-ag--galactosidase-fusion protein (B) in mechanically
perforated HTC cells in vitro. Each data point represents the
average of > 10 separate measurements for each of Fnu (nucleolar
fluorescence), Fn (nuclear fluorescence), Fc (cytoplasmic
fluorescence), and autofluorescence, with Fnu/c being the ratio of Fnu
to Fc, and Fn/c being the ratio of Fn to Fc, respectively, subsequent
to subtraction of autofluorescence. Data was fitted for the function
Fn/c(t) = Fn/c
(1 - e
) or
Fnu/c(t) = Fnu/c
(1 - e
)(23, 26) ,
the Fn/c
and Fnu/c
values for granzyme
being 2.55 and 10.3, respectively, with the linear regression values
greater than 0.95. The Fn/c
values for IAF-labeled
T-ag-
-galactosidase-fusion protein and the 20 and 70 kDa
FITC-dextrans were 8.5, 0.86, and 0.25, with linear regression values
of 0.99, 0.99, and 0.97, respectively.
Quantitative
analysis showed that nuclear accumulation (Fn/c) was maximally about
2.5-fold above that of the cytoplasm, while nucleolar accumulation
(Fnu/c) was a further 4-fold higher (Fig. 5A). The kinetics
of granzyme B nuclear transport were very rapid (t of about 3.3 min), with nucleolar accumulation reaching
equilibrium about 2 times more slowly (t
of
about 7.5 min) (Fig. 5A). The fact that accumulation in
the nucleus reached steady state before that in the nucleolus implied
that granzyme B was transported to the nucleus prior to further
transport to the nucleolus. The kinetics of nuclear accumulation of
granzyme B were much faster than those of the T-ag fusion protein,
which attained half-maximal nuclear accumulation at about 18.6 min (Fig. 5B).
The inability of granzyme B isoforms
greater than 32 kDa to enter the nucleus suggested that the type of
carbohydrate addition might affect nuclear localization. To examine
this possibility, 32-kDa granzyme B purified from human peripheral
blood mononuclear cells stimulated with interleukin-2 was treated with
endo H to remove high mannose carbohydrate and then examined for its
ability to bind YAC-1 nuclei (Fig. 6A). There was no
significant difference in the proportional uptake of endo H-treated
granzyme B (26 kDa, equivalent to the size of the protein backbone)
compared with the untreated 32-kDa form. Similarly, YAC-1 nuclei
exposed to whole YT cytoplasmic lysate took up only granzyme B that was
endo H-sensitive, while that remaining unbound additionally contained a
number of larger molecules that were endo H-resistant (Fig. 6B). Therefore, nuclear uptake was not dependent
on the presence of carbohydrate but could be inhibited by the addition
of carbohydrate in excess of 6 kDa.
Figure 6: Effect of endo H treatment on granzyme B localization. A, Western blot analysis, showing binding of 32-kDa granzyme B immunopurified from human interleukin-2-stimulated PBMC to isolated YAC-1 nuclei, either following treatment of the granzyme with endo H (+) or untreated(-) (see ``Materials and Methods''). Following incubation, nuclei were pelleted, and nuclear proteins were extracted and compared for granzyme B content (bound) with granzyme B that remained in the supernatant (unbound). The markers at left represent the migration of molecular mass standards in kDa. B, Western blot analysis, showing binding of endo H-sensitive 32-kDa granzyme B in YT cytoplasmic lysate to YAC-1 nuclei (NUC). These same species and additional, larger endo H-resistant species were present in the unbound fractions (CYT). The markers at left represent the migration of molecular mass standards in kDa.
As granzyme B is a basic
molecule, its nuclear uptake might be explained by overall
electrostatic attraction. The binding experiments with YAC-1 nuclei
were therefore repeated in lysis buffer over the pH range
8.0-10.0 (Fig. 7). Once again, there was no effect on the
relative proportion of bound granzyme B. Nuclear uptake at pH values
higher than 10.0 could not be examined because of granzyme B
insolubility (data not shown). The chymotrypsin-like serine proteases
expressed in mast cells are structurally related to granzymes and are
also very basic molecules(29, 30, 31) . The
net charge ((Arg + Lys) - (Asp + Glu)) of human
granzyme B at neutral pH, +15 ((31) ) is comparable with
that of mouse mast cell protease-5 (+12, (30) ). Met-ase
has a net charge of +14, and its activity is readily detected in
the nucleus (16) . We therefore purified and fractionated
resident mast cells from the peritoneal cavities of mice, and examined
the distribution of the mast cell-specific protease, mouse mast cell
protease-5, by immunoblotting (Fig. 8). The mast cells, which
were pelleted through a metrizamide step-gradient, demonstrated a
strong signal at 30 kDa for mouse mast cell protease-5 but only in
the cytoplasmic fraction. No mouse mast cell protease-5 was detected in
the buoyant cells, which were mainly macrophages and lymphocytes. This
result was consistent with our observation that only granzyme serine
proteases have a propensity for nuclear uptake.
Figure 7: Binding of purified granzyme B to YAC-1 nuclei at different pHs. Equal amounts of granzyme were added to nuclei at the pHs indicated, and the amount absorbed by the nuclei (N) or remaining unbound (S) was detected by probing with anti-granzyme B mAb in Western blot analysis.
Figure 8: Fractionation of mouse mast cell protease-5 in mouse mast cells. Western blot analysis, using anti-mouse mast cell protease-5 antiserum of nuclear (N) and cytoplasmic (C) lysates of resident mouse peritoneal mast cells pelleted through metrizamide (pellet). Equivalent numbers of macrophages/lymphocytes that were buoyant on metrizamide were fractionated simultaneously (buoyant). The markers at left represent the migration of molecular mass standards in kDa.
Granzymes are strong potential candidates for complementing the role of perforin in apoptosis. They share very similar patterns of tissue expression (32) and are co-packaged and co-released with perforin during degranulation (33, 34) and up-regulated by similar cytokine stimuli(35) . Despite the demonstrated synergy between perforin and granzyme B/fragmentin 2, several crucial issues remain open. A biochemical link is lacking between the proteolytic cleavage effected by granzyme B (at aspartate residues) and downstream target cell events that may lead to Cdc2 activation. A related question is the site of interaction of granzyme B with its putative substrate/ligand.
Our observations that granzymes,
but not non-granzyme serine proteases can be concentrated within nuclei
potentially have implications for the mechanisms of CTL/NK-mediated
cytolysis. The simplest model that would account for the collaboration
of perforin and granzyme B is that membrane fenestration induced by
perforin allows passive diffusion of granzyme B into the target cell,
enabling it to access a cytoplasmic or nuclear
ligand(5, 36) . To date, the presence of granzyme B in
the target cell cytoplasm has not been demonstrated. The only evidence
that granzyme B reaches the cytoplasm has been indirect, in that
protease inhibitors ``loaded'' into the cytoplasm of target
cells through the uptake of osmotically sensitive vesicles could
specifically block apoptosis(37) . We have recently used
immuno-gold staining and electron microscopy to demonstrate that
purified human granzyme B incubated with intact YAC-1 cells and
perforin is able to penetrate the YAC-1 cytoplasm within minutes. ()Our present observations imply that if granzymes are taken
up into the target cell cytoplasm, they may also be able to access the
nucleus. It is known that Cdc2 is translocated to the nucleus and
activated at the end of G
phase(38) . Furthermore,
the enzymes that influence the state of phosphorylation of Cdc2,
including the kinase Wee1 and the phosphatase Cdc25, do so at least
partly within the nucleus(15) . Therefore, factors that
influence cell cycle control through Cdc2 might impinge on nuclear
target molecules.
The degree of nuclear accumulation we observed is at least 10-fold greater than can be accounted for by simple diffusion, suggesting active granzyme B binding in the nucleus. Furthermore, sequestered granzymes retained their proteolytic activity within the nucleus. Following their synthesis on the endoplasmic reticulum and addition of high-mannose carbohydrate, nascent granzyme B molecules are targeted to the lysosome-like subcellular granules by binding to the mannose 6-phosphate receptor(39) . This pathway should allow concentration of granzymes within these specialized organelles, ready for release. Our previous observations that granzymes could be localized within the nuclei of cytolytic lymphocytes may be accounted for by the perturbation of granzyme processing and storage that follows cell disruption, and may reflect avid, specific granzyme uptake by the nuclei of these cells secondary to cell disruption. It is reasonable to assume that unlike cytolytic cells, most target cells lack the normal granzyme sorting pathways and that if free granzyme molecules can access the cytoplasm, sequestration within the nucleus might follow.
The mechanism by which granzyme B accumulates within the nucleus is
currently under investigation. Proteins smaller than 45 kDa may
diffuse into the nucleus through nuclear pores, and some of these are
actively transported out of the nucleus(40, 41) . In
contrast, larger proteins require an NLS that interacts directly with
the nuclear transport system in order to be imported into the
nucleus(27, 41, 42, 43) . We have
found that the forms of granzyme B greater than
32 kDa produced by
YT cells do not access the nucleus. This may either reflect a size
limitation or a specific effect of complex carbohydrate addition, e.g. masking of a crucial part of the granzyme molecule, which
is responsible for interaction with a nuclear structure or transport
molecule. Nucleolin, a protein that both shuttles between nucleus and
cytoplasm and is capable of binding NLS-carrying proteins (40, 44) , has previously been noted to bind to and
act as a substrate for granzyme A(45) . Granzyme A might use
nucleolin to enter the nucleus, as it is normally found as a 65-kDa
dimer, too large for simple diffusion. Alternatively, granzyme B may
have a functional NLS such as that of T-ag
(Pro-Lys-Lys-Lys-Arg-Lys-Val)(46) , sequences similar to which
have been identified in a number of other nuclear
proteins(41, 42, 43) . Granzymes are
generally basic proteins, and several stretches of basic residues could
serve as candidate NLSs.
Although we have directly visualized only granzyme B within nuclei and nucleoli, our biochemical data strongly suggest that granzyme A and Met-ase can also undergo nuclear uptake. The RBL transfection experiments of Henkart and co-workers (47) suggest that protease specificities other than Asp-ase are capable of inducing programmed cell death in collaboration with perforin. Furthermore, the cytotoxic activity of CTLs from granzyme B deficient mice was restored if the assay was prolonged(48) , implying that alternative granzymes could substitute for granzyme B, albeit with reduced efficiency. Granzymes might cleave nucleosomal proteins to bring about some of the nuclear changes associated with apoptosis and may not be required for initiating the apoptotic event per se. Cytoplasts that lack a nucleus are still susceptible to stimuli that produce programmed cell death(49, 50) . The role of a tryptase such as granzyme A might be to cleave Arg/Lys-rich histone proteins, thus permitting access of nucleases to DNA, consistent with our observation that active granzymes can be recovered from nuclei. Although a nucleus may not be required for apoptosis, an important function of granzymes might be to initiate destruction of virally contaminated DNA.
Recently, intriguing parallels have been drawn between the mammalian
enzyme, interleukin-1-converting enzyme (ICE) and the products of
cell death genes of primitive organisms such as the nematode Caenorhabditis elegans, with which it shares structural
similarity(51) . Like granzyme B, both ICE and the C.
elegans protein CED-3 are cysteine proteases with Asp-ase activity (52) . ICE can induce apoptosis when transfected into mammalian
cells (53) . The possibility that granzyme B might mimic the
actions of ICE or ICE-like molecules, either by direct activation, or
by cleaving a downstream substrate has been the cause of considerable
speculation. An ICE-like molecule, CPP32
(also termed Yama or
apopain), has recently been shown by several groups to cleave the
nuclear protein poly(ADP-ribose) polymerase at the sequence
Glu-Val-Asp-Gly
, while inhibition of the Asp-ase activity
of CPP32
abrogated
apoptosis(54, 55, 56, 57) .
Poly(ADP-ribose) polymerase cleavage is observed early in
apoptosis(58) . Poly(ADP-ribose) polymerase cleavage may be
blocked by CrmA, a serpin produced by the cowpox virus that inhibits
cell death(