©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Localization of Granzyme B in the Nucleus
A PUTATIVE ROLE IN THE MECHANISM OF CYTOTOXIC LYMPHOCYTE-MEDIATED APOPTOSIS (*)

(Received for publication, September 5, 1995; and in revised form, December 4, 1995)

Joseph A. Trapani(§)(¶) Kylie A. Browne Mark J. Smyth (§) David A. Jans (1)

From the Cellular Cytotoxicity Laboratory, The Austin Research Institute, Studley Road, Heidelberg, 3084, Australia, and the Nuclear Targeting Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, 2600, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

CTL (^1)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(2)/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.


MATERIALS AND METHODS

Cells and Cell Culture

The cell lines YT (human NK leukemia), rat basophilic leukemia (RBL), and HL-60 (human promyelocytic leukemia), were maintained in RPMI medium supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). YAC-1 (mouse lymphoma) and HTC (rat hepatoma) cells were maintained in Dulbecco's modified Eagle's medium supplemented as above. Resident mast cells were purified from the peritoneal cavities of BALB/c and DBA/2 mice as described previously(17) .

Extraction of Nuclear Proteins

Cells were washed 3 times in serum-free medium and lysed by resuspension in buffer containing 0.5% (v/v) Nonidet P-40, 25 mM KCl, 5 mM MgCl(2), 10 mM Tris-HCl, pH 7.4, on ice for 30 min. The nuclei were pelleted, and the cytoplasmic lysate (supernatant) was stored at -20 °C. The nuclei were resuspended in 10 ml of ice-cold Nonidet P-40 lysis buffer supplemented with 1% (v/v) Triton X-100, incubated on ice for 10 min, and then repelleted. The Triton X-100 extraction was then repeated. Nuclear proteins were extracted with high salt buffer (10 mM Tris-HCl, 500 mM NaCl, 0.1% Nonidet P-40, 5 mM ethylenediaminetetraacetic acid, pH 8.0), and insoluble debris pelleted by ultracentrifugation (100,000 times g for 20 min, 4 °C; Beckman model TL-100) and discarded.

Absorption of Granzyme B by Nuclei

Nuclei were prepared from RBL, HL-60, and YAC-1 cells as described above. Human granzyme B was immunopurified from YT nuclei as described elsewhere(16) . Nuclei (10^7, 25 µl packed volume) were mixed with either immunopurified granzyme B (10 µg) or unfractionated cytoplasmic lysate from YT cells in 1.0 ml of Nonidet P-40 lysis buffer whose pH was adjusted to 7.4, 8.0, 9.0, or 10.0 and incubated on ice for 30 min. The nuclei were pelleted and washed 3 times in 10 ml of Nonidet P-40 lysis buffer. All manipulations were performed strictly at 4 °C, using ice-cold buffers. Nuclear proteins were then extracted in 1.0 ml of high salt extraction buffer and analyzed by Western blotting or for protease activity, as described below.

Western Blotting

Western blotting was performed essentially as described previously(18) . The mAbs used were 2C5, a mouse IgG2a that reacts specifically with human and rat granzyme B(18) , and PB2, a mouse IgG1 reacting with human perforin(19) . An antiserum reactive with human BiP was purchased (StressGen, Victoria, Canada). Antiserum to mouse mast cell protease-5 was obtained from Dr. Patrick McNeil, Prince of Wales Hospital, Sydney, Australia.

Assay of Proteases

Modified microtiter assays were used to measure the granzyme activities of cytosolic and nuclear lysates. N-alpha-Benzyloxycarbonyl-L-lysine thiobenzylester esterase activity was estimated with a microtiter assay as described previously(20) , using purchased N-alpha-benzyloxycarbonyl-L-lysine thiobenzylester (Sigma). Thiobenzylester peptide substrates were used to measure cleavage after aspartic acid (Asp-ase) and methionine residues (Met-ase). The thiobenzyl esters Boc-Ala-Ala-Asp-S-benzyl and Boc-Ala-Ala-Met-S-benzyl were kindly provided by Dr. James Powers, School of Biochemistry, Georgia Institute of Technology, Atlanta, GA, and were used as described previously(21, 22) .

Endoglycosidase H (endo H) Treatment

Lysates and purified proteins were incubated with endo H (Boehringer Mannheim) for 60 min at ambient temperature, as specified by the manufacturer.

Fluorescein Labeling of Proteins

Immunoaffinity purification of human granzyme B from the nuclear extracts of YT cells was performed exactly as described previously(16) . The granzyme B was demonstrated to be free of granzyme A and Met-ase activities and perforin by Western blotting and functional assays (data not shown). For fluoresceination, granzyme B (60 µg/ml) or highly purified human chymotrypsin (60 µg/ml) (ICN Biochemicals) were equilibrated in 0.2 M NaCl, sodium borate buffer, pH 9.2. FITC (20 µl, 1 mg/ml in the same buffer) was then added for 2 h at ambient temperature. Uncoupled FITC was removed by dialysis in 0.1 M Tris-HCl, 0.1% NaN(3), 0.2 M NaCl, pH 7.4. Both the granzyme B and chymotrypsin retained 75% of their proteolytic activity under these reaction conditions. 5-Iodoacetamido fluorescein (IAF, Molecular Probes) was used to label bacterially expressed SV40 large tumor antigen (T-ag) fusion proteins as described previously(23, 24, 25, 26) .

Immunofluorescent Staining of Isolated Nuclei

Nuclei were isolated from YAC-1 cells, incubated with fluoresceinated human granzyme B and washed as described above and then centrifuged onto glass slides, using a cytocentrifuge (Cytospin 3, Shandon). The nuclei isolated in this way remained intact morphologically, and as judged by staining with propidium iodide (data not shown). The slides were air-dried for 1 h at ambient temperature, mounted with coverslips and visualized using a CLSM (Bio-Rad MRC-600) and a Nikon 60times Plan Ap oil immersion objective (numerical aperture, 1.4) as described previously (23, 24, 26) .

Nuclear Transport Assay

Nuclear transport was performed on mechanically perforated HTC cells essentially as described previously (23, 27) . Cells were grown on coverslips (15 mm times 15 mm) for 36 h to 50-70% confluency and then placed for 1 min in intracellular buffer (IB, 110 mM KCl, 5 mM NaHC0(3), 5 mM MgCl(2), 1 mM EGTA, 0.1 mM CaCl(2), 20 mM Hepes, pH 7.4, to which 1 mM dithiothreitol, and 10 µg/ml leupeptin were freshly added). Excess liquid was drained, IB (3 µl) was added to the cells, and then a single layer of tissue paper was placed onto the cell monolayer and rapidly removed 3 s later to mechanically perforate the cells, leaving the nuclei intact(27) . The coverslip was then placed onto a 5-µl drop of nuclear transport mix containing 45 mg/ml cytosolic extract (untreated reticulocyte lysate; Promega catalog no. L415A, aliquoted, and stored in liquid nitrogen at 150 mg/ml), an ATP regenerating system (0.125 mg/ml creatine kinase, 30 mM creatine-phosphate, 2 mM ATP), and transport substrate (30 µg/ml FITC-labeled granzyme B or chymotrypsin, 0.2 mg/ml IAF-labeled SV40 T-ag fusion protein, or FITC-labeled dextran). This mixture had been prepipetted into a chamber on a slide created by 1-mm-thick double-sided pressure-sensitive tape (Scotch 3MM) into which a hole of 0.8 mm in diameter was punched. The slide was finally sealed with nail polish to prevent dehydration during the incubation, which was generally for up to 1 h at room temperature.

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-beta-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) .


RESULTS

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.



Only 32-kDa or Smaller Forms of Granzyme B Are Accumulated within Nuclei

We next examined whether the nuclear uptake of granzyme B was dependent on its size. The NK cell line, YT, aberrantly expresses diversely glycosylated forms of granzyme B, consisting of a predominant 32-kDa molecule bearing high-mannose carbohydrate and larger forms up to 67 kDa that have more complex glycosylation(18) . All of these species can largely be reduced to a 26-kDa protein backbone by culturing the cells in medium containing tunicamycin(18) . Cytoplasmic lysate from YT cells was incubated with the nuclei of RBL, HL-60 or YAC-1 cells. In each case, the 32-kDa species was selectively bound by the nuclei (Fig. 2A). By contrast, other granzyme B forms (Fig. 2A) and perforin (Fig. 2B) remained totally in the supernatant. The nuclear fractions from YAC-1 were also analyzed for the chaperone protein BiP, which is abundantly found in the endoplasmic reticulum(28) . The nuclear fraction was devoid of BiP, demonstrating that the endoplasmic reticulum had been totally removed form nuclei used in this study. Therefore, granzymes were not simply adsorbed by residual endoplasmic reticulum adhering to the nuclear envelope.


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).



Subcellular Localization of Granzyme B

Since the subcellular fractionation data indicated nuclear association of granzyme B, immunopurified protein was fluoresceinated, incubated with YAC-1 nuclei, and centrifuged onto glass slides. The subcellular localization was then examined using CLSM (Fig. 3). All nuclei showed strong nucleoplasmic fluorescence, with the nucleoli being particularly bright (Fig. 3A). The results for quantitative analysis indicated that nucleolar fluorescence was over 2-fold that of the nucleus after the subtraction of autofluorescence (Fig. 3C).


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 beta-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-beta-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-beta-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(max)bullet(1 - e) or Fnu/c(t) = Fnu/c(max)bullet(1 - e)(23, 26) , the Fn/c(max) and Fnu/c(max) values for granzyme being 2.55 and 10.3, respectively, with the linear regression values greater than 0.95. The Fn/c(max) values for IAF-labeled T-ag-beta-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.




DISCUSSION

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. (^2)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(2) 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-1beta-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, CPP32beta (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 CPP32beta 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(