From the Granzyme B (GzmB) is a neutral serine protease
found in cytotoxic lymphocytes; this enzyme is critically involved in
delivering the rapid apoptotic signal to susceptible target cells. GzmB
has been difficult to study and has not yet been produced in
non-mammalian systems because of the complex processing events that are
thought to be required for its activation. In this report, we have
successfully produced fully active, soluble recombinant GzmB (rGzmB) in
a yeast-based system by fusing GzmB cDNA in frame with yeast
GzmB1 belongs to a
family of serine proteases specifically expressed in cytolytic T
lymphocytes (CTL), natural killer (NK) cells, and lymphokine-activated
killer (LAK) cells. The importance of GzmB for inducing target cell
death has been demonstrated via several lines of evidence (reviewed in
Refs. 1 and 2) and confirmed with GzmB null mutant mice (3). CTL and NK
cells derived from these mice have a severe defect in their ability to
induce early DNA fragmentation and apoptosis in susceptible target
cells. Furthermore, in vivo data suggest that GzmB plays a
significant role in the development of acute
graft-versus-host disease (4, 5). Therefore, a specific
inhibitor of GzmB may potentially prove to be an important tool in the
modulation of immune responses.
Among all the known granzymes, human and murine GzmB possess an unusual
preference for aspartic acid at the P1 site of its substrates (6). This
specificity is shared by only one other group of eukaryotic proteases,
the caspases (reviewed in Ref. 7). Experiments with cell-free systems
and caspase inhibitors have strongly implicated these enzymes in
apoptotic cell death (reviewed in Refs. 8 and 9). Caspases are
synthesized as proenzymes that generally require processing at aspartic
acid residues for activation. Although the enzymes responsible for the
activation of these caspases are unknown, there is evidence that these
enzymes can autoactivate or cross-activate other family members
(10-13). In vitro studies have also shown that GzmB can cleave and activate caspase-3 (CPP32) (14), caspase-8 (FLICE) (15), and
other caspases; in addition, GzmB-deficient CTL fail to cleave
caspase-3 in target cells during CTL-induced apoptosis (16). However,
these data have not determined whether GzmB induces apoptosis through
the activation of caspases or whether it acts directly to induce
apoptosis. Several studies have shown that peptide-based,
selective caspase inhibitors (which do not inhibit GzmB) can block
apoptotic cell death and target cell lysis induced by the
Fas-FasL pathway, but not the CTL granule exocytosis
(perforin/granzyme-dependent) pathway (17, 18).
GzmB is the prototype of a family of serine proteases (A-K) that are
synthesized as preproenzymes with an 18-residue leader peptide (23 residues for granzyme A) (reviewed in Ref. 19). These leader peptides
are thought to be cleaved by a signal peptidase, leaving a prodipeptide
that must be removed to produce the active enzyme (20). The enzyme
thought to be responsible for the specific cleavage of the prodipeptide
is the lysosomal cysteine protease DPPI (21-23). Previous studies in
COS cells have shown that co-transfection of a full-length (prepro)
human GzmB cDNA along with a rat DPPI cDNA resulted in the
generation of an enzymatically active GzmB, whereas transfection of
full-length GzmB cDNA alone led to the production of a
catalytically inactive enzyme (22). In addition, recombinant granzyme A
(rGzmA) can be converted into a proteolytically active enzyme by
incubation with bovine DPPI (23).
Although native GzmB (nGzmB) has been purified, only limited amounts of
relatively pure enzyme have been obtained. Previously, small amounts of
rGzmB have been successfully prepared from a mammalian COS
cell-based system (22, 24). However, the inability to purify sufficient
amounts of rGzmB (without any other contaminating cellular enzymes) has
limited the ability to study substrate specificities and to design
specific inhibitors. rGzmA has been successfully expressed in
Escherichia coli (25) and vaccinia virus (23); however, we
have repeatedly failed to generate soluble active rGzmB using a variety
of expression systems.
In this study, we describe the production of a yeast-based, soluble,
secreted form of active rGzmB. This recombinant protein has the same
specificities as nGzmB purified from an NK tumor cell line. We showed
that pro-GzmB (which contains the prodipeptide Gly-Glu) can be
converted to a fully active form by bovine DPPI. The active mature
rGzmB can cleave caspase-3 into its signature p20/p10 forms. We also
determined that several peptide-based caspase inhibitors do not
significantly inhibit the Asp'ase activity of rGzmB; furthermore,
Zn2+, a potent inhibitor of caspase-3 (26), fails to
inhibit rGzmB. These results underscore the fundamental differences in
substrate specificities between the caspases and GzmB.
Production of Mature and Pro-rGzmB in Pichia pastoris--
The
cDNAs encoding the pro and mature forms of murine GzmB were
amplified using the forward primers 5 Preparation of Native Murine GzmB--
Murine GzmB was isolated
from an NK-like tumor line derived from tumors that arose in transgenic
mice expressing SV40 large T-antigen driven by the human granzyme H
5 Western Blotting--
Western analysis was performed using the
polyclonal rabbit anti-murine GzmB antibody as described previously
(27). Equivalent amounts (0.5 µg) of pro-GzmB, rGzmB, or purified
nGzmB were used for each lane of an SDS-PAGE reducing protein gel under
reducing conditions. The gels were either stained directly with the
rapid silver stain kit (ICN Biomedicals, Aurora, OH) or transferred to
nitrocellulose and immunoblotted with immune sera at a 1:500 dilution,
as described previously (27).
Enzyme Assays--
Enzyme assays were performed using equivalent
amounts of pro-GzmB, mature rGzmB, and nGzmB as determined by
absorption at 280 nm and by Western blotting. Asp'ase and Met'ase
assays were carried out in a 500-µl volume using 3-5 µl of enzyme
(0.3-0.5 µg) in assay buffer containing 0.1 M Hepes, pH
7.5, 0.3 M NaCl, 1 mM EDTA, 0.11 mM
dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma), and 0.1 mM
substrate. Reactions were incubated at 23 °C for 30 min, and color
development was measured at A405 nm. Background (non-enzymatic) hydrolysis of substrates was assayed using 5 µl of
MEB buffer in the same assay buffer, including substrate and DTNB.
Tryptase assays were performed in a 200-µl volume using 0.3-0.5 µg
of enzyme in phosphate-buffered saline, pH 7.5, containing 0.22 mM DTNB, 0.20 mM
N- Determination of Substrate Kinetics--
Equivalent amounts of
rGzmB and nGzmB were defined by Western analysis. The initial rates of
hydrolysis were measured at 412 nm ( Endoglycosidase H (Endo H) Digestion--
Endo H treatment of
proteins to remove N-linked oligosaccharides was performed
in 100-µl reactions using 5 µl (0.5 µg) of pro-GzmB, rGzmB, or
nGzmB in an assay buffer containing 75 mM sodium citrate,
pH 5.5, and 5 µl of 1 unit/ml Endo H (Sigma). Negative controls were
performed using 5 µl of MEB buffer with or without Endo H. The
reactions were allowed to incubate overnight at 37 °C. Ten µl of
the reaction products were analyzed on SDS-PAGE gels and immunoblotted.
Fifty µl of the reaction products were used to assay for Asp'ase
activity with Boc-Ala-Ala-Asp-S-benzyl as the substrate.
Pro-GzmB Activation by DPPI--
5 µl (0.5 µg) of pro-GzmB
was incubated with 0.1-0.4 unit of purified bovine spleen DPPI (Sigma)
in 50 µl of assay buffer containing 50 mM sodium acetate,
pH 5.0, 30 mM NaCl, 1 mM EDTA at 37 °C
overnight. The reaction was diluted 10-fold in Asp'ase buffer, and
Asp'ase activity was measured as described above with Boc-Ala-Ala-Asp-S-benzyl as the substrate. The activity was
compared with the activity of equivalent amounts of active rGzmB. For
inhibition studies, DPPI was incubated with varying concentrations of
glycine-phenylalanine-diazomethylketone (G-F-CHN2) (29), a
potent and specific inhibitor of DPPI, for 2 h prior to the
addition of pro-GzmB. The reactions were then allowed to proceed
overnight as described above. To control for any direct effect of
G-F-CHN2 on Asp'ase activity, active rGzmB was also
incubated with G-F-CHN2 prior to the activity assay.
CPP32 in Vitro Translation and Activation--
mCPP32 cDNA
was generated by reverse transcription-polymerase chain reaction using
RNA derived from GzmB-deficient CTL effectors (3). RNA was prepared as
described (30). Approximately 1 µg of RNA was incubated with 200 ng
of oligo(dT)16 and 50 ng of random hexamers (Perkin-Elmer)
for 10 min at 68 °C. After 2 min on ice, 10 mM dNTPS,
100 mM dithiothreitol, 0.5 µl of SuperScript reverse
transcriptase (Life Technologies, Inc.) was added and incubated at
37 °C for an hour. After adjusting the total volume to 100 µl with
diethyl pyrocarbonate water, 1 µl of the first strand cDNA was
used as template. The 833-base pair cDNA was then amplified
using the forward primer 5 Department of Internal Medicine,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References
-factor cDNA, using the yeast KEX2 signal peptidase
to release the processed enzyme into the supernatant of yeast cultures.
We expressed the proenzyme form of GzmB as well and determined that
pro-GzmB is efficiently converted to its active form by the cysteine
proteinase dipeptidyl peptidase I. The fully processed enzyme was able
to hydrolyze the synthetic substrate
N-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat
of 17 s
1 and catalytic efficiency
kcat/Km of 181,237 M
1 s
1; the recombinant enzyme
is therefore at least twice as active as purified native GzmB. In
addition, the recombinant enzyme hydrolyzes Boc-Ala-Ala-Met thiobenzyl
ester with a kcat of 3.2 s
1 and a
catalytic efficiency
kcat/Km of 65,306 M
1 s
1. Purified rGzmB can also
cleave the putative substrate caspase-3 into its signature p20/p10
forms. Unlike caspases, rGzmB is not sensitive to inhibition by several
peptide-based inhibitors, including Ac-DEVD-CHO, Ac-YVAD-CMK, and
ZIETD-FMK, as well as Zn2+ (a known inhibitor of
caspase-3). Structural studies of rGzmB may allow us to better
understand the substrate specificity of this enzyme and to design
better inhibitors.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
-GGGGAGATCATCGGGGGGAC-3
(pro)
and 5
-ATCATCGGGGGACATGAAGC-3
(mature) and the reverse primer
5
-TTAGCTTTTCATTGTTTTCTTTATCCAGGATAAGAAACTTGAGAC-3
. A 5
XhoI site and a 3
EcoRI site were introduced
into the cDNAs and the products subcloned into the expression
vector pPIC9 (Invitrogen, Carlsbad, CA). Both inserts were completely
sequenced and found to be identical (except for the prodipeptide
Gly-Glu). Both vectors were electroporated into GS115 (Invitrogen) at
1.5 KV, 50 µF, and 186 ohms. His+ transformants were
selected and induced with methanol for 4 days according to a protocol
suggested by the manufacturer (Invitrogen). Daily aliquots of induced
supernatants from His+ clones were assayed for Asp'ase
activity and probed (using Western blotting) for the presence of rGzmB
using a polyclonal rabbit anti-mouse GzmB antibody (27). The clones
expressing pro-GzmB and mature rGzmB were selected and further
expanded. A liter of supernatant from each clone was precipitated using
85% ammonium sulfate, resuspended, and dialyzed to a final
concentration of 250 mM NaCl in 50 mM MES, 0.1 mM EGTA, 10% betaine (MEB buffer). The concentrated
supernatant was loaded onto a Mono S cation exchange column, and
proteins were eluted using a linear gradient of 0.25 M NaCl
to 1 M NaCl. Pro-GzmB and rGzmB both eluted at
approximately 0.6 M NaCl.
-flanking promoter sequence.2 This cell line was
used because it expresses abundant murine GzmB (28). Briefly, cells
were harvested, pelleted, resuspended in 1 M NaCl, 0.1%
Triton X-100, and sonicated. The crude extract was cleared by
centrifugation, dialyzed to a final concentration of 250 mM
NaCl in MEB buffer, and loaded onto a Mono S column. Proteins were
eluted with a linear gradient of 0.25 to 1 M NaCl. Native
murine GzmB eluted at approximately 0.6 M NaCl.
-benzylcarbonyl-L-lysine thiobenzyl ester (BLT), and 0.01% Triton X-100. Reactions were incubated at 37 °C for 20 min and stopped by adding 1 ml of 1 mM
phenylmethylsulfonyl fluoride; color development was measured at
A412 nm. Protease substrates included:
Boc-Ala-Ala-Asp-S-benzyl (Enzyme Systems Products, Dublin,
CA) for Asp'ase activity; Boc-Ala-Ala-Met-S-benzyl (Enzyme
Systems Products) for Met'ase activity;
Boc-Ala-Ala-Phe-S-benzyl (Enzyme Systems Products) for
chymase activity; Z-Lys-S-benzyl (Calbiochem) for
tryptase activity. Protease inhibitors included: Z-Ile-Glu-Thr-Asp-FMK (Enzyme Systems Products),
Ac-Asp-Glu-Val-Asp-CHO (Bachem, Torrance, CA), Ac-Tyr-Val-Ala-Asp-CMK
(Calbiochem), and 3,4-dichloroisocoumarin (Calbiochem). Stock solutions
of substrates and inhibitors were prepared in Me2SO and
stored at
70 °C.
412 = 14,150 M
1 cm
1) using a Cary 3 UV-visible spectrophotometer (Varian, Sugarland, TX) after 10 µl
(0.043 µM) of enzyme stock solution was added to a
cuvette containing 1.0 ml of buffer, 0.33 mM DTNB, and 40 µl of substrate (10-500 µM) at 20 °C. After
substraction for background hydrolysis, measurements were plotted using
a KaleidaGraph program (Synergy, Reading, PA), and the
Km and kcat for each substrate were determined using the Michaelis-Menten equation. Correlation coefficients for all plots were >0.99.
-GATCTCTAGAGGAACGCTAAGAAAAGTGACCATGG-3
(containing an XbaI site) and the reverse primer
5
-GATCAAGCTTCCTCTAGTGATAAAAGTACAGTTCTTTCG-3
(containing a
HindIII site) and subcloned into
XbaI-HindIII cleaved pBSKS (Stratagene, La Jolla,
CA).
/
CTL effectors. These extracts were generated and
prepared as described previously (27). After 2 h at 37 °C, the
samples were analyzed on a 10% SDS-PAGE reducing gel. The gel was then fixed, treated with Amplify (Amersham Corp.), and autoradiographed for
2 days.
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RESULTS AND DISCUSSION |
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P. pastoris is a methanotrophic yeast that has been
successfully used to produce large quantities of foreign proteins
(reviewed in Refs. 31 and 32). A major advantage of the P. pastoris expression system is that the foreign protein can be
secreted (along with very low levels of native proteins) as the first
step of purification. In addition, the secreted proteins are frequently correctly folded and not hyperglycosylated (33). We designed two GzmB
expression vectors for expression in Pichia: the first form included
the codons for the activation dipeptide Gly-Glu (pro-GzmB); the second
form starts with the Ile codon at the N terminus of the processed
enzyme (rGzmB) (see Fig. 1A).
These cDNAs were cloned in frame with the -factor signal peptide
in pPIC9, which targets them to the secretory pathway. The expressed proteins also contain the KEX2 recognition sequence
Glu-Lys-Arg directly preceding Gly-Glu (pro-GzmB) or Ile-Ile (rGzmB),
which allows signal peptide cleavage by the endogenous yeast peptidase KEX2 (34) (Fig. 1A). The supernatant from clones
that expressed cleaved pro-GzmB and rGzmB were purified on a Mono S
cation exchange column according to the "Experimental Procedures."
The major species detected on silver-stained gels (Fig. 1B)
and in Western analysis (Fig. 1C) (using reducing
conditions) had apparent molecular masses of 30 and 32 kDa. These two
bands represent different glycosylated isoforms of the proteins, since
Endo H treatment altered the migration of pro-GzmB and rGzmB (as well
as nGzmB purified from NK-like cell lines) to approximately 25 kDa
(Fig. 1C), the predicted molecular mass of the mature
protein. The deglycosylated forms of rGzmB and nGzmB retained full
activity toward thiobenzyl ester substrates (data not shown). The final
yield of purified rGzmB from 1 liter of yeast culture was approximately
1 mg.
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The activity of each recombinant protein was determined using
thiobenzyl ester substrates with the same amount of test enzyme, as
demonstrated by silver stain and Western analysis (Fig. 1, B
and C). An equivalent amount of partially purified nGzmB
served as positive control. Pro-GzmB had no measurable Asp'ase
activity (Table I and Fig. 4); in
contrast, rGmB was fully active. To further quantitate the catalytic
efficiency of our active recombinant enzyme, we determined
Michaelis-Menten parameters for two known synthetic thiobenzyl ester
substrates of nGzmB. rGzmB hydrolyzed Boc-Ala-Ala-Asp-S-benzyl with an apparent
kcat of 17 s1 and a
kcat/Km of 181,237 M
1 s
1 (Table I). The catalytic
efficiency of rGzmB is approximately twice that of nGzmB; these values
are similar to those reported in the literature by Odake et
al. (6) and Poe et al. (35). Similarly, rGzmA is more
active than native GzmA purified from granules (25).
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Previous studies could not determine whether the Met'ase activity
detected in purified GzmB preparations was due to GzmB itself or to
another unidentified granzyme that coeluted with GzmB (6). We
definitively established that rGzmB hydrolyzes
Boc-Ala-Ala-Met-S-benzyl with a kcat
of 3.2 s1 and a
kcat/Km of 65,306 M
1 s
1. Again, the catalytic
efficiency of rGzmB as a Met'ase is twice that of nGzmB. The cleavage
of Boc-Ala-Ala-Met-S-benzyl suggests that there may be GzmB
substrates with Met at the P1 position instead of an Asp. rGzmB has no
detectable tryptase or chymase activity (Table I), consistent with
previous reports (6).
To our knowledge, caspase-3 is the only caspase that has been shown to be a substrate of GzmB both in vitro as well as in vivo (12, 14). Caspase-3 presumably exists in all cells in its inactive, 32-kDa proenzyme form. During CTL-mediated attack on susceptible target cells, nGzmB processes caspase-3 to its active p20/p10 heterodimeric form (12, 36). For this reason, we wished to demonstrate that rGzmB is capable of recognizing and cleaving this physiological substrate. 35S-Labeled caspase-3 was cleaved to its signature p20 form by both nGzmB as well as rGzmB (Fig. 2, lanes 3 and 4), whereas neither pro-GzmB nor extracts from CTL effectors deficient in GzmB were capable of processing caspase-3 (Fig. 2, lanes 2 and 5).
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Previous studies have shown that two peptides (Ac-DEVD-CHO (37) and Ac-YVAD-CHO or Ac-YVAD-CMK (38, 39)) are potent inhibitors of caspase-3 and caspase-1, respectively. A recent study, using a combinatorial approach, defined the tetrapeptide IEPD as an optimal substrate sequence for GzmB (40). We therefore tested the ability of Ac-DEVD-CHO, Ac-YVAD-CMK, and ZIETD-FMK (a fluoromethyl ketone that closely matches the optimal substrate for GzmB) to inhibit rGzmB. We found that these peptides have essentially no inhibitory effect on rGzmB (or nGzmB) at concentrations up to 800 µM (Fig. 3). It should be noted that the methyl side chains added to ZIETD-FMK to enhance cell permeability might have affected the ability of this peptide to inhibit GzmB in vitro. Prolonged preincubation of the peptides with rGzmB (up to 4 h) did not increase the inhibition (data not shown). 3,4-Dichloroisocoumarin (DCI), a general serine protease inhibitor (4), inhibits rGzmB with an IC50 value of approximately 5 µM (Fig. 3). We also tested the inhibitory effect of Zn2+ (a potent inhibitor of caspase-3 with an IC50 of 0.1 µM (26)) and found that Zn2+ has no effect on rGzmB at concentations up to 300 µM. Other cations such as Cu2+, Mg2+, Mn2+, Fe3+, and Ca2+ also had no effect on the activity of rGzmB (data not shown).
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The ability of GzmB to cleave caspases, and the similarities between the specificities of GzmB and caspases (especially the requirements for Asp in the P1 position and Glu in the P4 position (40)), have suggested that GzmB is an integral part of the caspase cascade leading to cell death. However, more recent reports using specific caspase inhibitors suggest that GzmB may lead to target cell death through a caspase-independent pathway (16). These reports, and our results with peptide and cation inhibitors, underscore the differences between the two classes of enzymes. Although the sequence IEPD may be an optimal substrate for GzmB in vitro, specific inhibition of GzmB may require additional residues beyond the P4-P1 positions.
GzmB, along with the other cytotoxic lymphocyte granzyme family members, is synthesized as a preproenzyme with an 18 residue signal peptide followed by an activation dipeptide Gly-Glu. Activation requires processing of the signal peptide and the activation dipeptide. This dual proteolytic processing pathway appears to apply to the other granzymes, as well as structurally related serine proteases expressed in other hematopoietic cells such as neutrophil elastase, cathepsin G (21), and mast cell chymases (29). The enzyme involved in the proteolytic processing of the prodipeptide is thought to be the lysosomal cysteine protease DPPI (21-23, 29). Previous reports on the processing of rGzmB by purified bovine DPPI demonstrated specific (but low level) conversion to the active form of GzmB. Possible explanations include cross-species differences in substrate specificities (i.e. bovine DPPI processing human GzmB), inefficient subcellular colocalization of the two enzymes in transiently transfected COS cells or a requirement for an unidentified granule component to stabilize the proenzyme (20). We therefore tested the ability of bovine DPPI to process recombinant pro-GzmB to its active form in vitro. We found that at pH 5.0, bovine DPPI efficiently activated pro-GzmB; the degree of activation appears to depend only on the concentration of exogenous DPPI added (Fig. 4A). DPPI itself has no effect on the Asp'ase assay, nor does it affect the activity of mature rGzmB (Fig. 4A). This suggests that once the prodipeptide is removed, mature GzmB is resistant to further proteolytic cleavage by DPPI. The activation process is inhibited by the highly specific DPPI inhibitor Gly-Phe-CHN2 (29), at concentrations as low as 0.6 µM (Fig. 4B). These results, and previous reports (22-23), indicate that DPPI alone is sufficient for the activation of pro-GzmB. A null mutation of DPPI created by homologous recombination in the mouse should confirm the importance or redundancy of this enzyme for the activation of the granzymes and related proteins.
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In summary, this study is the first to demonstrate expression of active rGzmB in a non-mammalian system. The enzyme expressed has the same substrate specificities as the purified native enzyme. Inhibition studies also show that the current peptide-based inhibitors of caspases are ineffective at blocking the activity of GzmB. The Pichia system will allow us to produce large amounts of pure recombinant GzmB for structural studies that may allow us to better define substrate specificities and requirements for GzmB inhibition.
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ACKNOWLEDGEMENTS |
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We thank Dr. Linda Kurz and Marion Riley (Department of Biochemistry, Washington University) for their expertise and assistance in the determination of rGzmB kinetic constants. We also thank Drs. Tom Daly and Mark Sands for preparation of graphs included in the manuscript and Nancy Reidelberger for preparing the manuscript. We thank Dr. Steve Smeekins for suggesting that we try the Pichia system for the production of granzymes.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants K08 HL03774 (to C. T. N. P.) and DK 49786 and CA 49712 (to T. J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by the Buzz Fitzpatrick Memorial Fund of the Medical Research Council (Arlington, VA).
To whom correspondence should be addressed: Washington
University School of Medicine, 660 South Euclid, Box 8007, St. Louis, MO 63110. Tel.: 314-362-8831; Fax: 314-362-9333; E-mail:
timley{at}im.wustl.edu.
1 The abbreviations used are: GzmB, granzyme B; rGzmB, recombinant GzmB; nGzmB, native GzmB; CTL, cytolytic T lymphocytes; NK, natural killer; DPPI, dipeptidyl peptidase I; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; DTNB, dithiobis(2-nitrobenzoic acid); Boc, t-butoxycarbonyl; Endo H, endoglycosidase H; G-F-CHN2, glycine-phenylalanine-diazomethylketone; CMK, chloromethyl ketone; FMK, fluoromethyl ketone.
2 D. MacIvor and T. J. Ley, submitted for publication.
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REFERENCES |
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