(Received for publication, March 19, 1997, and in revised form, May 8, 1997)
From the Division of Bone Marrow Transplantation and Stem Cell Biology, Departments of Internal Medicine and Genetics, Washington University School of Medicine, Campus Box 8007, St. Louis, Missouri 63110-1093
Cytotoxic lymphocytes contain granules that have
the ability to induce apoptosis in susceptible target cells. The
granule contents include perforin, a pore-forming molecule, and several granzymes, including A and B, which are the most abundant serine proteases in these granules. Granzyme B-deficient cytotoxic T lymphocytes (CTL) have a severe defect in their ability to rapidly induce apoptosis in their targets, but have an intact late cytotoxicity pathway that is in part perforin-dependent. In this report,
we have created mice that are deficient for granzyme A and
characterized their phenotype. These mice have normal growth and
development and normal lymphocyte development, activation, and
proliferation. Granzyme A-deficient CTL have a small but reproducible
defect in their ability to induce 51Cr and
125I-UdR release from susceptible allogeneic target cells.
Since other granzyme A-like tryptases could potentially account for the
residual cytotoxicity in granzyme A-deficient CTL, we cloned the murine
granzyme K gene, which is linked to granzyme A in humans, and proved
that it is also tightly linked with murine granzyme A. The murine
granzyme K gene (which encodes a tryptase similar to granzyme A) is
expressed at much lower levels than granzyme A in CTL and LAK cells,
but its expression is unaltered in granzyme A/
mice. The minimal
cytotoxic defect in granzyme A
/
CTL could be due to the existence
of an intact, functional early killing pathway (granzyme B dependent),
or to the persistent expression of additional granzyme tryptases like
granzyme K.
The cytolytic granules of cytotoxic T lymphocytes (CTL)1 and natural killer (NK) cells contain several different neutral serine proteases called granzymes, the pore-forming protein perforin, and other proteins of unknown function. The granule exocytosis model of cellular cytotoxicity postulates that granzymes, in concert with perforin, induce target cell apoptosis after cytotoxic lymphocytes specifically recognize the target cell, and release their granule contents into the intercellular space between the effector and target cells (1). Perforin is thought to produce channels in the target cell membrane through which the granzymes pass; in the target cell, granzymes are thought to cleave and activate critical substrates, thereby initiating the apoptotic program of the cell. Several distinct granzymes from mice, rats, and humans have been cloned and characterized. To date, granzymes A through G and metase-1 have been identified in mice (2-13), granzymes A, B, K, and metase-1 in rats (14-19), and granzymes A, B, H, K, and metase-1 in humans (20-29). Except for granzyme B, which is required for the rapid induction of target cell apoptosis during CTL attack (30-32), the precise functions of the other granzymes are not yet known.
Granzymes A and B are the most abundantly expressed granzymes found in
CTL stimulated with a wide variety of activation protocols. Granzyme A
is a tryptase (which cleaves after lysine or arginine at the P1
position) and granzyme B is an Asp'ase (which cleaves after aspartic
acid at the P1 position). The genes encoding these proteases have been
mapped to two different chromosomal loci: 1) murine and human granzyme
A are found on chromosome 13 and a syntenic region of human chromosome
5, respectively (23, 33, 34), and 2) both murine and human granzyme B
are found on syntenic regions of chromosome 14 (35-37). The granzyme B
gene is part of a cluster of tightly linked granzymes and other serine
protease genes that are specifically regulated and expressed in
distinct hematopoietic cell types. The murine granzyme B locus contains granzymes B-G, cathepsin G, and MMCP-2 (and probably other mast cell
chymases); the human granzyme B locus is known to contain granzymes B
and H, cathepsin G, and mast cell chymase. Our laboratory has
demonstrated that the insertion of PGK-Neo cassette in the murine
granzyme B gene disrupts the expression of granzymes C, D, F, and G in
LAK/NK cells, potentially confounding interpretations of some of the
results obtained from these mice (38). Specifically, the defect
observed in the ability of granzyme B/
LAK cells to induce
apoptosis of susceptible targets could be due in part to the loss of
granzyme C, D, F, or G. However, CTL derived from MLR predominantly
express granzymes A and B. Thus, in our studies of granzyme B
/
CTL,
the defect in the rapid induction of apoptosis is most likely due to
the loss of granzyme B itself.
Studies of granzyme B/
mice have revealed that CTL are equipped
with a late, granzyme B-independent mechanism(s) of cytotoxicity (30,
31). Several lines of evidence implicate granzyme A as a potential
candidate for this pathway. Specifically, 1) purified granzyme A from
mouse CTL or rat NK granules induces apoptosis of
detergent-permeabilized or perforin-permeabilized target cells (16,
39); 2) noncytotoxic rat basophilic leukemia cells transfected with
perforin and granzyme vectors induce DNA fragmentation in target cells
only when they express both perforin and granzyme A or B
(40, 41); and 3) granzyme A is a "late" acting protease, since NK
granule-purified granzyme A induces apoptosis only after 14 h of
incubation time, compared with 2 h of incubation time needed for
the production of granzyme B-induced apoptosis (16).
Another tryptase called granzyme K is also a candidate for granzyme B-independent cytotoxicity. The murine orthologue of human granzyme K (28, 29) has not yet been identified, but rat granzyme K has been purified and its cDNA has been cloned (16, 18). Similar to granzyme A, granzyme K purified from a rat large granular lymphoma cell line (RNK-16) induces apoptosis of target cells in a late 14-h cytotoxicity assay (16). Moreover, human granzymes A and K map to the same location on chromosome 5q11-q12 (42). These data suggested that murine granzyme K is likely to exist, and that it may be located near the granzyme A gene on mouse chromosome 13.
In this paper, we report the cloning of murine granzyme K and establish
the tight linkage between murine granzymes A and K. We have created a
null mutation of granzyme A by replacing exon 2 and a significant
portion of intron 2 with a standard PGK-Hygro cassette. We have found
that our granzyme A/
mice are viable and have normal development,
fertility, and hematopoiesis, similar to the mice reported by Ebnet
et al. (43), which bear a different mutation in the granzyme
A gene. Expression of the granzyme K gene is not altered by the
insertion of the PGK-Hygro cassette in the granzyme A locus. Granzyme
A
/
CTL have only a slightly reduced ability to attack and kill
allogeneic target cells, perhaps because they have an intact granzyme B
cluster, and/or perhaps because they exhibit persistent expression of
granzyme K (and/or D), which is also a potent tryptase.
Total RNAs
derived from mouse splenocytes activated with concanavalin A/IL-2 for 2 days, or from an NK-like cell line (a CD3, NK1.1+ splenic tumor line
harvested from transgenic mice expressing simian virus 40 (SV40) T
antigen driven by the human granzyme H
promoter)2 were isolated
using a previously described method (44). The first strand of cDNA
was synthesized with reverse transcriptase (Life Technologies, Inc.)
and random hexamer primers. Using KlenTaqLA (Wayne Barnes, Washington
University, St. Louis), double-stranded cDNA templates were then
PCR amplified with the following pair of primers that were derived from
rat sequences and corresponded to the most conserved regions between
the human and rat granzyme K cDNAs: 5
-CATTCCAGGCCTTTTATGGC-3
and
5
-GGATCAAAAGCAAGCTTGCC-3
. A 650-bp PCR product was subcloned into the
pCR 2.0 vector (Invitrogen, San Diego). The inserts were sequenced with
an ABI373 automated sequencer.
A 500-bp murine granzyme A probe spanning the two alternatively spliced leader exons of granzyme A was generated using PCR amplification of mouse genomic DNA. The PCR amplified probe was sequenced and tested in Southern analysis to confirm its ability to detect unique mouse granzyme A bands. This probe was then used to screen a murine BAC library (Genome Systems, St. Louis, MO). Four BAC clones (BAC 1254-1257) that hybridized strongly with the granzyme A fragment were obtained and subsequently tested for the presence of granzyme K gene in Southern analysis using the 650-bp murine granzyme K cDNA as the probe. One BAC clone, BAC 1255, hybridized with the granzyme K cDNA probe. DNA obtained from BAC 1255 was then digested with HindIII or PstI, and two HindIII and one PstI fragments were subcloned into pUC19 based vectors and sequenced.
Mapping of the Distance between Murine Granzymes A and K LociDNA isolated from BAC 1255 was digested with rare cutting
restriction endonucleases, electrophoresed on 1% agarose gels using a
CHEF DRII apparatus (Bio-Rad), transferred onto Hybond (Amersham) membranes, and probed with either the 500-bp 5 murine granzyme A probe
or a 2.0-kb murine granzyme K HindIII genomic fragment containing exons 1 and 2. The hybridizing and washing conditions of
these Southern blots were performed as described previously (37).
Murine splenocytes were activated either with concanavalin A/IL-2, or with one-way mixed lymphocyte reactions (MLR) as described previously (30). Mouse spleen cells were also cultured with high dose (1000 units/ml) recombinant human IL-2 for 10 days to generate LAK cells, as described previously (31).
Analysis of mRNATotal cellular RNA was isolated from
activated lymphocytes and analyzed using an S1 nuclease protection
assay, as described (45). Probes specific for murine granzyme A, murine
granzyme B, and murine 2-microglobulin genes were
previously reported (30). A probe that specifically detected correctly
spliced exon 4 of murine granzyme K was created using a 1.8-kb
BglII genomic fragment isolated from the plasmid containing
the 4.7-kb HindIII genomic fragment of granzyme K. Correctly
spliced granzyme K mRNA protects a probe fragment of 119 nucleotides from S1 digestion. All probes were labeled to approximately
the same specific activity.
The
500-bp murine granzyme A DNA probe that spans the alternatively spliced
leader exons was used to screen a 129/Sv mouse genomic library
(Stratagene). A clone containing an 8-kb BamHI fragment
containing most of the murine granzyme A gene was obtained. The 8-kb
genomic fragment was subcloned into pUC9, and Southern blot and partial
sequencing analyses confirmed that this DNA contained the 5
-flanking
region and exons 1 through 4 of mouse granzyme A. This 8-kb
BamHI fragment was used to generate the granzyme A gene
targeting vector. A 2.7-kb EcoRI-HindIII region
containing exon 2 of the granzyme A gene was replaced with a 1.6-kb
fragment containing the hygromycin phosphotransferase gene driven by
the phosphoglycerate kinase I gene promoter (PGK-hygro (46)), resulting in an exon 2 deletion mutation in the granzyme A gene. The PGK-hygro cassette was inserted in the opposite transcriptional orientation from
granzyme A. The completed targeting vector consisted of a 3.5-kb
BamHI-EcoRI genomic fragment containing
5
-flanking sequence and leader exons upstream from PGK-hygro, and a
1.9-kb HindIII-BamHI genomic fragment including
exons 3 and 4 downstream from PGK-Hygro.
RW4 ES cells
(derived from 129/SvJ mice) were transfected with the granzyme A
targeting vector, and hygromycin-resistant clones were isolated
essentially as described (30). The resistant ES clones were screened by
Southern blotting using a 327-bp 3 external probe that was PCR
amplified with primers based on published mouse granzyme A genomic
sequences (47, 48). The external probe detected a 9-kb wild-type and a
5-kb mutant allele of PstI-digested mouse genomic DNA. Two
out of 110 ES clones (ES 64 and ES 144) were correctly targeted, and
these two clones were further confirmed in a Southern analysis with a
hygromycin phosphotransferase specific probe. C57BL/6 blastocysts were
then microinjected with ES cells from each of the two targeted clones
and implanted into pseudopregnant Swiss Webster foster females, as
described previously (30). Chimeric males with a high percentage of
agouti color were mated with C57BL/6 females, and their offspring were
examined for germline transmission of the disrupted granzyme A allele
by Southern analysis using the 3
external probe described above. ES 64 derived heterozygotes were intercrossed to produce homozygous mutant
mice.
Thymocytes, splenocytes, and activated splenocytes from day 5 MLRs were stained with fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies against various mouse cell surface markers and analyzed using a FACScan flow cytometer and CellQuest version 1.2.2 software (Becton-Dickinson), as described previously (30).
BLT Esterase Activity Assay1 × 107 CTL
were resuspended in 100 µl of 1 × Hebs buffer (137 mM NaCl, 5.0 mM KCl, 0.7 mM
Na2HPO4, 6.0 mM dextrose, and 20 mM Hepes, pH 7.05) and 100 µl of 2 × lysis buffer
(2 M NaCl, 50 mM Tris, pH 7.5, and 0.2% Triton
X-100), sonicated for 2-3 s, and centrifuged at 4 °C for 5 min at
10,000 rpm to clear the supernatant from cellular debris. 20 µl of
this supernatant was tested for tryptase activity using the BLT
(N-benzyloxycarbonyl-L-lysine
thiobenzyl ester) esterase (Calbiochem) activity assay as described
previously (49). Briefly, the test sample was mixed with 200 µl of
BLT substrate solution (phosphate-buffered saline with 0.2 mM BLT, 0.22 mM 5,5
-dithiobis(2-nitrobenzoic acid) (Pierce), and 0.01% Triton X-100). After the reaction mixture was incubated at room temperature for 30 min, 1 ml of
phosphate-buffered saline solution containing 1 mM
phenylmethylsulfonyl fluoride (Sigma) was added to stop the reaction.
The OD410 was then measured using a Beckman DU-20
spectrophotometer. Controls included the test sample and substrate
solution without BLT (phosphate-buffered saline with 0.22 mM 5,5
-dithiobis(2-nitrobenzoic acid) and 0.01% Triton
X-100) or the BLT substrate solution alone.
Standard 51Cr or 125I-UdR release assays were performed exactly as described (50).
To identify the mouse
homologue of human and rat granzyme K, we performed reverse
transcriptase-PCR using primers based on rat cDNA sequences that
corresponded to the most conserved regions between the human and rat
(77% identity between human and rat granzyme K cDNAs). We
amplified a 650-bp product from Con/IL-2 activated mouse splenocytes
and a mouse NK-like cell line. Sequence analysis confirmed that the
650-bp fragment from each cDNA source contained a highly homologous
cDNA encompassing exons 2, 3, 4, and most of 5. Fig.
1 shows the deduced amino acid sequence
of this cDNA. The 650-bp cDNA sequence isolated was not
full-length, but the 5 region, all of exons 1 through 5, and a
putative polyadenylation signal (AATAAA) were identified in the genomic
sequence (Fig. 2).
The mouse granzyme K cDNA, which is predicted to encode 264 amino acids, is 89.0 versus 77.6% identical to the rat and human cDNAs, respectively. Based on previous work, the NH2 terminus of the mature, active protease is designated +1 at the isoleucine position shown in Fig. 1. The deduced amino acid sequence of murine granzyme K shares 86.0 versus 70.9% identity with the rat and human homologues, respectively. In the aligned amino acid sequences of the mouse, rat, and human granzyme K, the catalytic triad residues (His41, Asp90, and Ser188) and six of eight cysteine residues that are expected to form three internal disulfide bonds (26-42, 123-155, and 173-194) are conserved among all three species (Fig. 1). The two remaining cysteine residues (184 and 208) may form a fourth disulfide bond. The granzyme K sequence does not contain any consensus sites for N-linked glycosylation.
Among the murine granzymes, granzyme K cDNA is most similar to granzyme A (53.5% identity). Granzymes A, K, and other tryptases have an aspartic acid residue at position 182 (six residues amino-terminal to the active site serine) that is thought to determine specificity for lysine or arginine in the P1 position. However, in contrast to granzyme A (which forms a 60-kDa homodimer due to an interchain bond made by the ninth cysteine) granzyme K probably acts as a monomer because it does not contain the additional cysteine residue. Therefore, the active, mature murine granzyme K protein is predicted to be a 26-kDa monomer, in keeping with the size of purified rat (16, 18) and human granzyme K (24).
Genomic Organization and Linkage of the Granzyme K and A Genes in MiceWe screened a mouse BAC library derived from 129/SvJ mice
using a 500-bp genomic fragment containing the alternatively spliced leader exons of mouse granzyme A, and obtained four clones (BAC 1254-1257) that hybridized with this probe. Southern analysis of DNA
from the BAC clones (using probes encompassing various regions of
murine granzyme A locus) confirmed the presence of the entire granzyme
A genomic sequence in all four BAC clones. These clones were further
examined by Southern blotting using a 650-bp murine granzyme K cDNA
probe spanning exons 2 through 5; one clone, BAC 1255, hybridized
strongly with this probe. A HindIII or PstI
digest of BAC 1255 DNA probed with the 650-bp granzyme K cDNA probe
revealed two HindIII (2.0 and 4.7 kb) and one
PstI (2.2 kb) fragment that were subcloned into pUC 19 vectors. All three plasmids were completely sequenced using
oligonucleotides based on the cDNA and the newly derived genomic
sequences. The 2.0-kb HindIII fragment contained the 5
region and exons 1 and 2; the 4.7-kb HindIII piece contained
exons 3, 4, and most of 5; and the 2.2-kb PstI fragment
included exons 4 and 5 and 3
-flanking sequence. The complete genomic
structure of mouse granzyme K is shown in Fig. 2. Consensus intron
donor and acceptor sites are present in the expected position (Fig.
2A). The entire gene consists of 5 exons and 4 introns (Fig.
2B), a common feature for this family of genes. Granzyme A
spans over 10 kb and contains two alternatively spliced leader exons
(47, 48); the granzyme K gene encompasses ~6 kb of genomic DNA, and
appears to have only one leader-encoding exon.
Since the localization of both granzymes A and K to BAC 1255 indicated
that the two genes are closely linked, we next attempted to map the
precise distance between them. DNA from BAC 1255 was treated with
rare-cutting restriction enzymes and analyzed using Southern blotting
with mouse granzyme A or K-specific probes. The granzyme A-specific
probe consisted of a 500-bp DNA fragment that spans the
alternatively spliced exons (described above). The 2.0-kb
HindIII granzyme K probe contained the 5 region and exons 1 and 2, and detected unique granzyme K bands in Southern blots of mouse
genomic DNA. SunI, AscI, SgfI, and
PacI all cleaved the insert once; NotI released
the insert of 145 kb, which contained both genes. However, a 65-kb
PmeI fragment of BAC 1255 DNA hybridized to both the
granzyme A and K probes, revealing that the two genes are at most 65 kb
apart (data not shown). None of the co-digests with enzymes that cut
once or twice within the insert localized the two genes on a smaller
fragment, so the precise distance between the two genes is not yet
resolved.
We investigated the expression pattern of murine granzyme K
mRNA by S1 nuclease protection analysis, using a granzyme
K-specific probe end-labeled in exon 4 (Fig.
3). Correctly spliced granzyme K mRNA
protected an exon 4-derived fragment of 119 nucleotides from S1
digestion. A tissue survey revealed no detectable granzyme K mRNA
in murine bone marrow, heart, kidney, liver, lung, small intestine,
large intestine, resting spleen, or thymus (data not shown). We next
examined granzyme K mRNA levels in day 5 MLR-derived CTL, and in
LAK cells generated in splenocyte cultures activated with 1000 units/ml
IL-2 for 10 days. In addition to the exon 4 granzyme K probe, probes
for granzyme A and 2-microglobulin were used as controls
for lymphocyte activation, and for RNA quality and content,
respectively. The results in Fig. 3 show the presence of granzyme K
mRNA, along with the expected expression of granzyme A and B
mRNAs, in both MLR-derived CTL and LAK cells. Densitometric analysis of these autoradiographs revealed that granzyme K mRNA levels are ~1.5% that of granzyme A in day 5 MLR cells, and ~15% that of granzyme A in LAK cells.
Creation of Mice with a Targeted Disruption of the Granzyme A Gene
We generated granzyme A/
mice using the targeting vector
shown in Fig. 4A (top
panel). The mutant, homologous recombinant granzyme A allele
contains a deletion of exon 2 (which contain the histidine residue of
the catalytic triad). Using a 3
probe that lies just outside of the
sequence used for targeting (Fig. 4A, middle panel), two
correctly targeted 129/SvJ-derived RW4 ES clones (out of 110 screened)
were identified. Both ES clones were injected into C57BL/6J
blastocysts. One clone gave rise to highly chimeric males that
transmitted the mutant granzyme A locus in the germline. In Fig.
4B, which shows a Southern analysis of PstI-digested genomic tail DNA of offspring generated by
mating the mutant granzyme A heterozygotes, the expected 9.0-kb wild type and 5.0-kb targeted granzyme A alleles were detected by the 3
probe described above. Heterozygote matings yielded mice with approximately Mendelian ratios of 40:59:34 (+/+:+/
:
/
) mice. Granzyme A+/
and
/
mice have normal development and fertility, and are grossly indistinguishable from their wild type
counterparts.
Lack of Granzyme A Expression in Granzyme A
S1 protection and BLT esterase activity assays were
performed with day 5 MLR-derived CTL and/or LAK cells generated from
granzyme A+/+ or /
mouse spleens. S1 protection analysis, using a
previously described exon 4-specific granzyme A probe (which lies
outside of the mutant region), revealed a complete absence of granzyme A mRNA in granzyme A
/
MLR-derived CTL (Fig.
5A). Granzyme A
/
cytotoxic
effector cells revealed a normal ability to activate in response to
allogeneic stimuli (MLR) or to IL-2 in LAK cell cultures, since
equivalent levels of granzyme B mRNA were detected in granzyme A+/+
and
/
CTL. Unaltered levels of granzyme K mRNA were detected in
the A
/
mice; the presence of the PGK-hygro cassette in the granzyme
A gene therefore does not reduce the expression of the closely linked
granzyme K gene.
We next measured the ability of MLR-derived granzyme A+/+ or /
CTL
to cleave the synthetic substrate BLT. Fig. 5B shows that a
markedly reduced amount of BLT is hydrolyzed by lysates from granzyme
A-deficient CTL; however, a small amount of residual BLT esterase
activity remains in these mutant CTL.
We measured the subsets of lymphocytes in
the thymus and spleen of granzyme A/
mice. Fig.
6 is a flow-cytometric analysis of
granzyme A+/+ or
/
lymphocyte populations stained with mAbs against
murine CD3, CD4, CD8, B220, and NK1.1 cell surface molecules. Granzyme
A+/+ or
/
thymocytes included the typically predominant population
of immature CD4+CD8+ (double-positive) cells and small percentages of
mature CD4+ or CD8+ (single positive) cells (Fig. 6A).
Similarly, both wild type and mutant mice had virtually identical proportions of all expected subsets of immune cells in their spleens (Fig. 6B). Day 5 MLR-derived granzyme A+/+ and
/
CTL
((H-2b) anti-Balb/c (H-2d)) had equivalent
percentages of CD3+, CD4+, and CD8+ cells, and the majority of these
CTL were CD8+, as expected (Fig. 6C). Moreover, cell counts
revealed similar numbers of wild type and mutant CTL generated in MLR
(data not shown), suggesting that granzyme A
/
splenocytes
proliferate normally. Together, the cell counts and FACS profiles imply
that granzyme A-deficient T lymphocytes develop normally and undergo
proper activation upon allogeneic stimulus in vitro.
Slightly Reduced Cytotoxic Activity of Granzyme A-deficient CTL against Allogeneic Targets
The normal proliferation and
activation of granzyme A/
splenocytes in response to allo-stimulus
in vitro allowed us to specifically address the ability of
the mutant, day 5 MLR-derived CTL to attack allogeneic targets using
standard lytic assays. Fig. 7 shows
results of 51Cr release and 125I-UdR release
assays at varying effector to target (E:T) ratios at an early (2 h) or
a late (8 h) time after the incubation of effector and target cells
together. In control experiments, both the wild type and mutant CTL
lacked cytotoxic activity against the syngeneic, H-2b
expressing EL4 targets at all time points and E:T ratios tested (data
not shown). Granzyme A
/
CTL (H-2b
anti-H-2d) were able to induce nearly normal levels of
51Cr or 125I-UdR release from the
H-2d expressing allogeneic TA3 (Fig. 7A), P815
(Fig. 7B), or YAC-1 (Fig. 7C) target cells at all
E:T ratios tested (at both early and late time points). The slightly
reduced levels of 51Cr and 125I-UdR release
mediated by the mutant CTL were consistently observed in multiple
experiments against a wide variety of target cells, including L1210
cells transfected with a sense or antisense Fas cDNA construct
(data not shown). Although the differences shown are small, they are
consistent and statistically significant.
Reduced 51Cr release and
125I-UdR release mediated by granzyme A/
CTL against
allogeneic targets. Standard 2- and 8-h lytic assays were
performed with day 5 MLR-derived granzyme A+/+ (squares) or
/
(circles) CTL against TA3 (panel A), P815
(panel B), and YAC-1 (panel C) allo targets at
varying E:T ratios. At both early (2 h) and late (8 h) time points, granzyme A
/
CTL induce
slightly less 51Cr release and 125I-UdR release
from all three target cell lines. This defect in cytotoxicity is
greatest at high E:T (30:1) ratios, and it is statistically significant
(p < 0.05) for the majority of data points. Values at
each point represent the mean of duplicate samples ± S.E. This
experiment is one of three with similar results.
In this report, we have described the phenotype of granzyme A/
mice obtained by gene targeting. Similar to granzyme A
/
mice
created by Ebnet and colleagues (43), our granzyme A-deficient animals
have normal development and fertility. Cytotoxic lymphocytes derived
from these mice have normal programs of activation and proliferation,
and express normal levels of the tightly linked granzyme K gene. In
addition, standard lytic assays performed with allo-specific CTL
derived from these mice reveal a small defect in their ability to
induce 51Cr and 125I-UdR release from
allogeneic target cells at all time points and E:T ratios tested. The
initial analysis of our granzyme A
/
mice therefore corroborates the
phenotype observed in cytotoxic effector cells derived from granzyme
A-deficient mice produced by Ebnet and colleagues (43).
The small defect in cytotoxicity present in granzyme A-deficient mice
is puzzling, since granzyme A is known to be an enzyme that is capable
of inducing apoptosis in target cells. However, the similar phenotypes
observed in the mice described by Ebnet et al. (43) and in
this report strongly suggest that the minimal phenotype is, in fact,
correct. The mice described by Ebnet et al. (43) contain an
insertional mutation of PGK-Neo in exon 4; this mutant gene could
potentially be alternatively spliced to produce mutant granzyme A
molecules that contain exons 1, 2, 3, and 5. However, no granzyme A
protein could be detected by Western analysis in that study, and
minimal residual tryptase activity was noted on BLT esterase studies.
No defect in the cytotoxicity of granzyme A/
CTL was observed, but
these mice failed to clear the cytopathic pox virus ectromelia in
normal fashion (51). In contrast, our mutation removes one of the exons
that contains part of the catalytic triad, and eliminates nearly the
entire amino terminus of the protein. A small amount of residual
tryptase activity was detected in the activated CTL from our mice; a
definite small reduction in cytotoxicity was observed, in contrast to
the normal cytotoxicity detected in the Ebnet et al. (43)
mice. The mice made by Ebnet et al. (43) are in an inbred
C57BL/6 background, and our mice are in a mixed BL6 × 129 background. Therefore, two different mutations in this gene
(created in two different genetic backgrounds) have yielded similar
phenotypes, making it extremely likely that these results represent an
accurate view of the granzyme A null mutant state.
There are several potential reasons why granzyme A/
CTL may have
minimally reduced cytotoxicity in vitro. First, granzyme A
/
mice have a functioning granzyme B locus. In previous work from
our laboratory, we have shown that enzymes in the granzyme B cluster
are required for the rapid induction of apoptosis in susceptible target
cells (30, 31, 38). Since the rapid killing pathway is intact in
granzyme A-deficient animals, the granzyme A defect may be nearly
impossible to detect. By creating animals that are doubly deficient for
granzymes A and B, we may be able to precisely define the role of
granzyme A for the late, granzyme B-independent cytotoxicity that we
have previously described (30, 31, 52). Careful analysis of the
cytotoxic phenotypes of these animals will also allow us to determine
whether any other molecules in cytolytic granules contribute
significantly to "perforin-dependent" cytotoxicity. If
granzyme A × B cluster-deficient animals have the same phenotype
as those that are perforin-deficient, then it would seem unlikely that
additional granule proteins contribute significantly to
granule-mediated cytotoxicity. Experiments designed to test these
hypotheses are in progress.
An alternative explanation for the minimal cytotoxic defect of granzyme
A deficient mice may be the "rescue" of granzyme A function by
other granzymes with a similar specificity. As noted above, granzyme K
is a potent tryptase. Despite its close linkage with granzyme A, the
PGK-hygro cassette does not alter granzyme K expression in granzyme
A/
CTL. In addition to granzyme K, granzyme D is another known CTL
tryptase (8), and its expression is unaltered in granzyme A-deficient
mice as well (data not shown), since it lies in the granzyme B locus on
chromosome 14. Regardless, granzymes D and K must contribute only
minimally to the total tryptase activity present in activated CTL,
since BLT esterase activity was reduced by more than 80% in our
granzyme A-deficient mice. However, the formal possibility exists that
the low level of residual tryptase activity provided by granzymes D, K,
and perhaps other granzymes is sufficient to rescue the mice from the
consequences of granzyme A deficiency.
As an initial step toward understanding the contribution of these other granzymes for CTL function, we have cloned the murine granzyme K gene which encodes a tryptase that is similar to granzyme A. Granzyme K has three structural features that are common to all serine proteases (Fig. 1): 1) the mature, active protease contains the characteristic NH2-terminal sequence (IIGG); 2) the catalytic triad residues (H, D, and S) are present at homologous positions flanked by conserved peptides; and 3) six cysteine residues (out of a total of eight) are conserved in homologous positions and are expected to form internal disulfide bonds. Of all the known granzymes with these common features, granzyme K is most similar to granzyme A. Both granzymes A and K have the same specificity-determining aspartic acid residue located six residues amino-terminal to the active site serine. Other substrate specificity defining amino acids, including glycine at position 205 and 209, are also conserved in identical positions in granzyme K. Besides these common tryptase determining residues, the two extra cysteines (which are not characteristic of all serine proteases) are located in homologous positions in granzymes A and K. These common structural features suggest that these two proteases may cleave similar physiological substrates and have similar functions. However, granzyme K does not contain the ninth cysteine residue that allows granzyme A to form homodimers. Furthermore, there are several stretches of amino acid residues that are conserved in granzyme K but not in granzyme A, suggesting that granzyme K could have slightly different substrate specificities and/or functions from granzyme A.
Highly related serine protease genes tend to occur in clusters (e.g. the "granzyme B" cluster and the "neutrophil elastase" cluster). Extensive studies with both the mouse and human granzyme B gene clusters have demonstrated that some genes within this cluster are expressed specifically in the activated lymphocyte compartment (35-37). Our present study, together with previously published work from the human system, establishes that granzymes A and K genes are closely linked as well. In the human system, Fink and colleagues (34) have previously demonstrated that the granzyme A gene maps to 5q11-12, a region that is syntenic between human chromosome 5 and mouse chromosome 13, band D, where the mouse granzyme A gene is localized. Moreover, using fluorescent in situ hybridization, Baker et al. (42) have shown that the human granzyme K also maps to 5q11-12, thus identifying a new locus of the tryptase family of serine proteases. In our study, we extend these findings by identifying both murine granzymes A and K genes in a 65-kb mouse genomic fragment derived from a BAC clone. The significance of the clustering of these genes is not yet clear, but it suggests that these genes share common regulatory elements, such as locus control regions.
Previous work has suggested that the expression of granzymes A and K is restricted to activated T cells and NK cells. Our results indicate that day 5 MLR-derived CTL and LAK cells express much lower levels of granzyme K than granzyme A mRNA. This observation corroborates data obtained by Hameed and colleagues (24), who have purified both granzymes A and K from IL-2 activated human LAK cells; they found that granzyme K protein is much less abundant than granzyme A. However, in that study, granzyme K had twice the specific activity of granzyme A. Rat granzyme K has been demonstrated to be a highly expressed granzyme that can be easily purified from RNK-16, the rat large granular lymphocyte leukemia cell line (16, 18); Shi and colleagues (16) have demonstrated that granzyme K is more abundant than granzyme A in RNK-16 cells. Further analysis of various activated cytotoxic lymphocyte populations is needed to more accurately determine the relative abundance of granzyme A versus K in cytotoxic lymphocytes, and to more precisely define the roles of these granzymes in cell mediated cytotoxicity.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF011446.
We thank Nancy Reidelberger for excellent secretarial assistance and Drs. Christine T. N. Pham and Timothy A. Graubert for their critical review of this manuscript.