(Received for publication, September 20, 1995; and in revised form, January 9, 1996)
From the
Granzyme B (cytotoxic cell proteinase 1) is a serine proteinase that has been implicated in cytotoxic T lymphocyte-induced apoptosis. In order to understand how granzyme B is involved in mechanisms of target cell destruction, characterization and identification of substrates are required. We have developed an in situ binding assay using permeabilized cells and recombinant granzyme B that allows us to visualize potential substrates after immunostaining with anti-granzyme B antiserum.
Confocal laser scanning microscopy and immunoelectron microscopic analyses demonstrate that granzyme B recognizes a nuclear substrate. The labeling pattern observed corresponds with regions of positive staining with uranyl acetate which binds to heterochromatin in the nucleus. Positive labeling of target cells with granzyme B is dependent on the presence of a catalytically active proteinase, since an inactive proenzyme form of granzyme B fails to give rise to any binding in the target cells. Far-Western blotting and immunoprecipitation of subcellular fractions of target cells have shown that the putative substrate of catalytically active granzyme B is an 80-kDa nuclear protein. Minor cytosolic bands of 50 and 94 kDa are also observed. A cytoplasmic band of 69 kDa is detected by both active and zymogen forms of granzyme B.
Apoptosis induced in target cells by cytotoxic T lymphocytes is
believed to occur after CTL()-target conjugation which is
facilitated by engagement of the T-cell receptor and its accessory
molecules(1) . This cell-cell contact induces the exocytosis,
from the CTL, of granules which contain the potential cytolytic
effector molecules. These include the pore-forming protein perforin
(cytolysin) and a family of serine proteases collectively known as
granzymes. The series of events that occur after degranulation, which
lead to target cell death, remain to be elucidated.
Perforin is believed to create pores in the target cell membrane which may disrupt osmoregulation and facilitate transfer of the granzymes. Once the granzymes enter a target cell, it is hypothesized that cleavage of their respective substrates results in cell death(2) . Hallmarks of CTL-induced cell death include many of the signs of apoptosis: DNA fragmentation, chromatin condensation, membrane blebbing, and cell shrinkage(3) .
Granzyme B has been
implicated in a number of these processes, most notably with the
fragmentation of DNA. Granzyme B is the prototypic member of this
family of proteases (4) and is most abundantly expressed in
response to a diverse array of CTL stimuli: -CD3, ConA, phorbol
12-myristate 13-acetate, and allogeneic stimulation(5) .
Expression of granzyme B correlates with lytic activity of CTL in
vitro(5) and with the activity of infiltrating T
lymphocytes in allografts in vivo(6) . Granzyme B has
also been isolated as the activity known as fragmentin(7) .
This activity is necessary for degradation of DNA in target cells by
CTL. A role for granzyme B in the degradation of target cell DNA is
further supported by the granzyme B homozygous null mutant
mice(8) . These mice have a severely depressed ability to cause
rapid DNA fragmentation in target cells.
Granzyme B has an uncommon
substrate specificity of aspartic acid at P1 (9, 10) .
This preference is also observed in ced3(11) and
interleukin 1-converting enzyme(12) . These molecules play
a pivotal role in apoptosis in Caenorhabditis elegans and
higher eukaryotes, respectively. Physiological substrates of this group
of enzymes with the rare substrate specificity of Asp at P1 will
undoubtedly provide insights into the respective apoptotic processes in
which they are involved. In this study, we present evidence to
demonstrate that catalytically active granzyme B will recognize a
nuclear protein that co-localizes with heterochromatin in target cells.
Catalytically active granzyme B and inactive proenzyme cloned in the vector pAX142 were expressed by transient transfection of COS M5 cells as described previously(14) . Enzymatic activity of mature granzyme B was assayed by cleavage of the synthetic substrate tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester in the presence of the chromogenic indicator 5,5`-dithiobis(2-nitrobenzoic acid) as described(14) .
Granzyme B was used in a far-Western
blot to screen for potential substrates. Protein blots of whole cell
lysates and subcellular fractions of target cell protein were analyzed
in this manner. Fig. 1shows that granzyme B binds to a nuclear
protein of M = 80,000 in all cell lines
examined. This protein is not recognized by inactive granzyme B
zymogen, which differs from the mature proteinase by an additional
dipeptide (Gly-Glu) at the N terminus. Minor cytosolic proteins of M
= 50,000 and 94,000 are also detectable (Fig. 2). A cytoplasmic protein of M
= 69,000 is recognized by both active and zymogen forms of
granzyme B (Fig. 2).
Figure 1:
Far-Western blot of nuclear extracts
labeled with granzyme B and zymogen. Electroblot of nuclear extracts
(50 µg/lane) labeled sequentially with granzyme B or zymogen,
-CCP, and donkey anti-rabbit IgG-horseradish peroxidase. Detection
is a result of chemiluminescence with ECL. A single band of
approximately 80-kDa is detected in all cell lines tested. Blots were
probed first with zymogen, stripped, and rescreened with active
granzyme B.
Figure 2:
Far-Western blot of EL4 subcellular
fractions labeled with granzyme B or zymogen. Each lane contains 50
µg of the indicated protein lysate. Detection is by
chemiluminescence as in Fig. 1. A nuclear band of 80 kDa is
detected with active granzyme B, as are faint cytosolic bands of 50 and
94 kDa. This signal is dependent on the addition of COS M5 lysate
containing either of these granzyme B species. Screening with -CCP
antiserum alone does not give rise to any 69-kDa bands(13) .
Granzyme B and zymogen both recognize a cytoplasmic protein of 69
kDa.
These observations are supported by
proteinase-linked immunoprecipitation of target cell proteins. EL4
proteins were fixed in 1% formaldehyde prior to incubation with
granzyme B and precipitated with -CCP antiserum and protein
A-Sepharose. Fig. 3shows an 80-kDa nuclear protein and 50-,
69-, and 94-kDa cytoplasmic proteins following purification by
protease-linked immunoprecipitation. Each band was subsequently
isolated by elution from SDS-polyacrylamide gel electrophoresis.
Treatment of the target cell proteins with formaldehyde is a necessary
measure to ensure the precipitation of these proteins, but renders the
proteins inaccessible to the sequencing chemistry.
Figure 3:
Proteinase-linked immunoprecipitation of
[S]Met-labeled EL4 protein lysates. Nuclear and
cytoplasmic fractions were incubated sequentially in 1% formaldehyde
and COS M5 lysate containing active granzyme B followed by
precipitation with
-CCP and Sepharose CL-4B-conjugated protein A.
Following immunoprecipitation, individual proteins were purified by
elution from SDS-polyacrylamide gel electrophoresis. A nuclear protein
of 80 kDa is precipitated (lane A) as well as cytoplasmic
proteins of 94, 69, and 50 kDa (lanes B-D,
respectively). The 69-kDa band is also precipitable with inactive
granzyme B zymogen.
In all
cell lines examined, approximately 50 to 80% of cells displayed uptake
of label and all cells that labeled demonstrate similar labeling
patterns. Fig. 4shows representative cells labeled with
granzyme B or zymogen. P815 target cells also show a nuclear labeling
with granzyme B. However, this pattern appears to be heavily
concentrated in the nucleolus (Fig. 4A). Endogenous
serine proteinases can be detected with the -CCP antiserum in a
cytoplasmic granular distribution, but this labeling pattern is not
shown in order to highlight the proteinase-specific nucleolar labeling
of P815 as a target cell. Permeabilization of cells with 0.1% saponin
was necessary to observe any signal arising from the addition of
granzyme B or
-CCP.
Figure 4:
In situ proteinase-linked
immunocytochemical analysis of granzyme B binding in target cells. P815 (A and D), L1210 (B and E), and EL4 (C and F) were labeled with granzyme B (A, B, and C) or zymogen (D, E, and F) followed by -CCP and Texas Red-conjugated goat
anti-rabbit IgG. All panels show cells counterstained with
FITC-conjugated ConA. Cells were viewed by
CLSM.
Granzyme B gives rise to a staining patterns in EL4, L1210 (Fig. 4B), and Jurkat (Fig. 5) that is granular throughout the nucleoplasm. This staining is consistent with nucleolar patterns observed in multinucleolar cell types and lines(17, 18, 19) . Labeling of these cells with granzyme B is observed throughout the cytoplasm, but cannot be attributed to a specific subcytoplasmic localization. Closer scrutiny of localization of granzyme B binding proteins in EL4 cells is described in immunoelectron microscopic studies below.
Figure 5: Nucleolar localization of granzyme B label in target cells. Jurkat cells were labeled with granzyme B (A) or zymogen (B) as described in Fig. 2, but using the nucleolar specific antisera ANA-N (FITC) as the secondary label. Granzyme B label appears red, ANA-N appears green, and coincidental label appears yellow. Cells were viewed by CLSM.
Double label of Jurkat cells with the nucleolar specific antiserum ANA-N and granzyme B shows a co-localization pattern. In these experiments, granzyme B binding appears red and the nucleolus is labeled green. Where the two patterns overlap, the label appears yellow. Fig. 5shows that murine granzyme B gives rise to a nucleolar labeling pattern in the human Jurkat target cell line. The Jurkat nucleoli appear green when labeled with the inactive zymogen, due to a positive green signal from the ANA-N and a lack of a red signal from granzyme B zymogen.
The importance of the negative control utilizing the zymogen granzyme includes other relevant negative controls for successful application of this system. Positive signals caused by interaction of the antisera binding directly to antigens in the target cells are ruled out, as well as any signals that may have arisen from the interaction of any of the secondary labeled antibodies to target cell proteins.
We first performed the far-Western blots with either active enzyme or zymogen. This was followed by stripping of the blots and rescreening with zymogen or active enzyme, respectively. This removed the possibility that the stripping process adversely affected the ability of either active enzyme or zymogen to bind proteins in this far-Western blot arrangement.
An additional measure
to demonstrate that the positive signals attributed to binding of
catalytically active granzyme B also represents a specific interaction
between the -CCP antiserum, and the granzyme B from the COS lysate
is the observation that preincubation of this antiserum with a bovine
serum albumin-conjugated octapeptide (the peptide that served as the
original immunogen) successfully competes out binding of the antiserum
to granzyme B COS lysate-labeled target cells.
Figure 6:
Proteinase-linked immunoelectron
microscopic analysis of granzyme B label of target cells. Ultrathin
sections of EL4 target cells were labeled with granzyme B (A)
or zymogen (B) in combination with -CCP and goat
anti-rabbit Ig gold conjugate (10 nm particle). Sections were
counterstained with uranyl acetate and lead citrate. Insets correspond to
2.5 magnification of the boxed area in each respective panel.
Cytoplasmic labeling by granzyme B observed by CLSM is not observed by electron microscopy. The glutaraldehyde fixative used for electron microscopy masks the binding of granzyme B to any potential cytoplasmic substrates. This masking effect was also observed when glutaraldehyde-fixed cells were viewed by confocal microscopy.
Far-Western blot analysis of EL4 subcellular fractions demonstrate that catalytically active granzyme B binds to a nuclear protein with an apparent molecular weight of 80,000 and cytosolic proteins of 50,000 and 94,000 that are not detected by inactive zymogen. This suggests that the interaction of granzyme B with these proteins is dependent on an active catalytic site within the proteinase facilitated through an open specificity pocket. A cytoplasmic protein of 69-kDa is recognized by both active and inactive forms of granzyme B. The possibility of a relationship between these proteins has not been examined. Granzyme B does not contain any known linear or bipartite nuclear localization signal, so the possibility exists that granzyme B requires a cytoplasmic protein to shuttle the proteinase into the nucleus. Perhaps the 69-kDa protein fills this role since a shuttle protein is not likely to be dependent on an active catalytic site in granzyme B.
Nuclear labeling with granzyme B shows a punctate staining pattern throughout the nucleus and is heavily concentrated surrounding or within the nucleolus. The nature of the nuclear labeling is dependent on the target cell line. Given that granzyme B recognizes 80-kDa nuclear proteins in all of the cell lines tested, this is likely to reflect differences in the nucleolar composition and distribution between cell lines rather than differences in the substrate(s) being recognized.
A more detailed nuclear labeling pattern was observed through protease-linked immunoelectron microscopy. These studies demonstrate that this nuclear granzyme B-binding protein co-localizes with the heterochromatin. This chromatin fraction contains transcriptionally inactive DNA, including the centromere which provides a link to the structural framework of the nuclear matrix. Also contained in the heterochromatin is DNA that is merely transiently transcriptionally repressed. The heterochromatin labeling of target cells with granzyme B is not likely to have arisen from recognition of a centromere-associated protein. Distribution of the centromere gives rise to a speckled staining pattern throughout the nucleus (20) and is markedly different from all patterns observed with granzyme B labeling.
One of the hallmarks of apoptosis is nucleolar disintegration (21) followed by DNA fragmentation and chromatin condensation(3) . Based on the data presented here, a hypothesis suggests that granzyme B may cleave a substrate that is responsible for maintaining the condensation of heterochromatin. Such a cleavage may then result in the exposure of a DNase-sensitive site or motif that would otherwise be sequestered. In this model, granzyme B would bring about the destruction of a target cell by rendering the target cell sensitive to destructive machinery that is constitutively present and active in the target cell.
These studies demonstrate that enzymatically active granzyme B can be used to successfully locate proteins within a target cell that are bound by granzyme B in a manner resembling proteinase-substrate interactions. Although granzyme A has been demonstrated to bind nucleolin in target cell lysates(22) , this is the first example of a granule serine proteinase from a CTL binding to proteins within a target cell in situ. The labeling pattern and the size of the proteins detected suggest that the 80-kDa protein recognized by granzyme B does not correspond to nucleolin (110 kDa) (23) which, although found in the nucleolus, displays a different nuclear labeling pattern(24, 25) .
Granzyme B has been demonstrated
to bind to four proteins in target cells. While transfer of granule
proteinases from a CTL to its respective target cell remains to be
visually demonstrated, these data show that granzyme B can recognize at
least two cellular proteins in the target cell. It has recently been
shown that granzyme B can cleave the interleukin 1-converting
enzyme-like protease CPP32(26) . Failure to detect CPP32 in
far-Western blots is likely due to the low abundance of CPP32. Given
the lack of sensitivity of the assay described here, the 50-, 69-, 80-,
and 94-kDa proteins are likely present in significant abundance.
Since the time period between the initial contact between CTL and target cell and ultimate destruction of the target cell is as low as 6 min(27) , it is quite likely that the effects of the CTL are mediated through a pathway that utilizes existing components within the target. Included in this observation is the probability that substrates of granzyme B are widely and constitutively expressed in all cell types that are sensitive to destruction by CTL. This hypothesis is supported by the observation of an 80-kDa granzyme B-reactive protein in a diverse panel of cell lines. The binding of granzyme B to these proteins may represent a proteolytic cleavage event that results in either the release of a destructive activity or renders some vital sequestered motif accessible to pre-existing cellular degradative processes.