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(Received for publication, November 17, 1994; and in revised form, January 4, 1995) From the
Trypomastigotes, the infective stages of the intracellular
parasite Trypanosoma cruzi, induce rapid and repetitive
cytosolic free Ca
Invasion of mammalian cells by intracellular pathogens is likely
to involve complex mechanisms of attachment, communication, and
internalization. Until recently, attention was focused primarily on the
identification of ligands and receptors which mediate binding. Although
many organisms gain access to the intracellular environment through
phagocytosis, some are capable of becoming internalized by cells with
little or no phagocytic capacity(1) . It has therefore become
apparent that pathogen factors, not necessarily exposed on the surface,
may be produced with the purpose of transducing signals to host cells
and changing their behavior. Several examples already exist of
signaling events and associated cellular changes that are likely to
facilitate entry of invading
pathogens(2, 3, 4, 5, 6, 7, 8, 9) . The invasion of nonphagocytic cells by the intracellular parasite Trypanosoma cruzi occurs by a unique mechanism. Unlike most
well studied intracellular pathogens, T. cruzi entry is not
blocked, but is facilitated by inhibitors of actin polymerization such
as cytochalasin D(10, 11) . Host cell lysosomes are
recruited to the site of parasite attachment, and gradual fusion with
the plasma membrane takes place during invasion(11) . Lysosome
fusion is thought to provide membrane for the developing vacuole which
surrounds the invading parasite for a short time after
entry(12, 13, 14) . This novel mechanism
for invasion would require communication between parasite and host cell
in the form of intracellular signaling. It was recently demonstrated
that trypomastigotes, the infective stage of T. cruzi, trigger
rapid, repetitive increases in the cytosolic free calcium concentration
([Ca The
present study was undertaken to characterize the trypomastigote factor
mediating Ca
Figure 1:
Total soluble extract of T. cruzi trypomastigotes induces elevation in
[Ca
Figure 2:
Titration of
[Ca
The characteristics of an
average response of NRK cells to 500 µl of trypomastigote total
soluble fraction (10
Figure 3:
Inhibition of parasite-induced
[Ca
As
a control for nonspecific interference of the protease inhibitors in
the spectrofluorometric assay, thrombin (0.2 unit/ml) was preincubated
with several of the protease inhibitors under the conditions described
in Fig. 3. These treatments had no inhibitory effect on the
Ca
No Z-F-R-AMC hydrolyzing activity was detected in assays
performed with intact NRK cells. Furthermore, preincubation of the NRK
cells with the irreversible inhibitor Z-F-R-FMK did not block the
Ca
Figure 4:
SDS-PAGE analysis of T. cruzi soluble proteolytic activity. Soluble extracts of T. cruzi trypomastigotes were separated by electrophoresis on 10% SDS-PAGE
gels (gel in panel A contained 0.1% co-polymerized gelatin).
The total soluble extract (T), ConA unbound fraction (UB), or ConA-binding fraction (GP) was loaded on
gels at 5
To
demonstrate that the 120-kDa peptidase exhibited the same sensitivity
to inhibitors of the Ca
Figure 5:
Comparison of peptidase and
Ca
Two additional conditions were
found in which the presence of an active 120-kDa enzyme did not
correlate with Ca
Figure 6:
Interaction with heparin and NEM inhibits
Ca
Figure 7:
Effect of protease inhibitors on invasion
of NRK cells by T. cruzi. Trypomastigotes were preincubated in
Ringer's solution (Control) or in Ringer's
solution containing the indicated concentrations of Ac-R-R-CMK or
Z-F-R-FMK and then added to NRK cells for 20 min at 37 °C. Cells
were washed and fixed, and the number of intracellular parasites was
determined by
immunofluorescence(11) .
We have provided, for the first time, evidence that an
intracellular parasite employs proteolytic processing to generate a
factor with Ca The majority of
the trypomastigote Ca Our results indicate that
proteolytic activity is tightly coupled to
Ca Ca The
characteristics of the 120-kDa enzyme appear to be identical with an
unusual T. cruzi alkaline peptidase described previously (16, 17, 18) including its low sensitivity to
E-64. This peptidase, which is expressed in all life cycle stages of T. cruzi(16) , cleaves substrates preferably on the
carboxyl side of arginine or lysine residues and has been suggested to
fulfill a processing role (17) . The 120-kDa enzyme appears to
have unique properties. As well as being unable to cleave any of the
large macromolecular substrates tested, it exhibited unusual inhibitor
sensitivity(17, 18) . While several inhibitors of
cysteine proteases inhibited the 120-kDa, the enzyme was not
significantly activated by thiol-containing reagents, nor was it
inhibited by E-64 or NEM. Cystatin C, an inhibitor of papain-like
proteases(26) , also failed to inhibit the enzyme (Table 2). Our results also show for the first time that the
120-kDa peptidase does not associate with the lectin concanavalin A,
indicating a possible lack of glycosylation. Clarification of this
issue and others regarding the active site of this peptidase will be
possible only when sequence information becomes available. Several
lines of evidence suggest that activity of the 120-kDa peptidase,
although required, is not sufficient to induce
[Ca The notion that T. cruzi proteases play a role in host cell invasion, differentiation, and
intracellular development of the parasite is not
new(28, 29, 32) . However, in previous
studies, cruzain has been proposed as the target for the effects of
permeable irreversible cysteine protease inhibitors including
Z-F-R-FMK. Our results demonstrate that Z-F-R-FMK and Ac-R-R-CMK, which
inhibit both the 120-kDa peptidase and Ca Proteolytic processing of prohormones in eukaryotic cells
is a common means of generating biologically active
molecules(34) . The enzymes responsible for processing cleave
at specific sites on the substrate, most often the C-terminal side of
single or paired basic residues. The preference of the 120-kDa enzyme
for the carboxyl side of basic residues suggests a strict substrate
specificity and thus is a good candidate for a processing enzyme as
previously suggested(17) . Based on data presented in this
paper, we propose that a trypomastigote-specific substrate is cleaved
by the 120-kDa peptidase and that this processing event is required for
the generation of a factor capable of inducing
[Ca Although the trypomastigote
substrate for the 120-kDa peptidase has not been identified, our
results suggest that it interacts with heparin and is sensitive to
alkylation by NEM. Binding to heparin is a property exhibited by
several growth factors(35) . The Ca Recent evidence indicates that communication between the parasite
and the host cell plays a role in invasion by T.
cruzi(11, 15) . Interaction of trypomastigotes
with the surface of host cells results in rapid and repetitive
increases in host cell [Ca The
present study demonstrates that an unusual parasite protease, the
120-kDa alkaline peptidase of T. cruzi, is involved in the
generation of a novel Ca
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5172-5180
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-signaling Factor for
Mammalian Cells (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
transients in fibroblasts.
Buffering or depletion of intracellular free Ca inhibits cell entry by trypomastigotes, indicating a role for
this signaling event in invasion. We show here that the majority of the
Ca-signaling activity is associated with the soluble
fraction of parasites disrupted by sonication. Distinct cell types from
different species are responsive to this soluble factor, and
intracellular free Ca transients occur rapidly and
reach concentrations comparable to responses induced by thrombin and
bombesin. The Ca-signaling activity does not bind
concanavalin A and is strongly inhibited by a specific subset of
protease inhibitors. The only detectable protease in the fractions with
Ca-signaling activity is an unusual alkaline
peptidase of 120 kDa, to which no function had been previously
assigned. The activity of the protease and cell invasion by
trypomastigotes are blocked by the same specific inhibitors that impair
Ca-signaling, suggesting that the enzyme is required
for generating the response leading to infection. We demonstrate that
the 120-kDa peptidase is not sufficient for triggering
Ca-signaling, possibly being involved in the
processing of precursors present only in infective trypomastigotes.
These findings indicate a biological function for a previously
identified unusual protozoan protease and provide the first example of
a proteolytically generated parasite factor with characteristics of a
mammalian hormone.
]
) in host cells,
whereas the noninvasive epimastigote stage does not(15) .
Fibroblasts respond to live parasites and subcellular fractions of
trypomastigotes in a pertussis toxin-sensitive manner, and trypsin
treatment of the active fraction impairs its ability to mediate a
Ca response. It was also observed that inhibition of
the [Ca] transients
interferes with host cell invasion by the parasite(15) .-signaling in host cells. We have found
that the majority of the Ca-signaling activity could
be recovered in a soluble form after parasite disruption, allowing us
to utilize a quantitative fluorometric assay to analyze the responses
of mammalian cells to this factor. The generation of the
[Ca] transients proved
to be dependent on the activity of a trypanosome alkaline peptidase
previously described (16, 17, 18) . Since the
alkaline peptidase itself has no Ca-signaling
activity, we propose that its role is to process a precursor present
only in the infective trypomastigote, generating a short-lived
hormone-like factor with the ability to trigger rapid increases in the
[Ca] of mammalian
cells.
Materials
Reagents were obtained from the
following sources: fura-2/AM and pluronic F-127 were obtained from
Molecular Probes, Inc. Methyl 
-D-mannopyranoside, methyl
-D-glucopyranoside, probenecid, EGTA, leupeptin,
aprotinin, chymostatin, soybean trypsin inhibitor (SBTI), (
)phenylmethylsulfonyl fluoride, dithiothreitol (DTT),
heparin, heparin-agarose, and gelatin were purchased from Sigma. trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane
(E-64) and N-ethylmaleimide (NEM) were obtained from
Boehringer Mannheim. Ionomycin was purchased from Calbiochem, and
concanavalin A-Sepharose from Pharmacia Chemicals (Uppsala, Sweden).
The synthetic peptides Z-Phe-Arg-7-amino-4-methylcoumarin (Z-F-R-AMC)
and Z-Gly-Gly-Arg-AMC (Z-G-G-R-AMC) were obtained from Enzyme Systems
Products (Livermore, CA). Z-Phe-Arg-fluoromethyl ketone (Z-F-R-FMK) was
provided by J. McKerrow (Veterans Administration Medical Center, San
Francisco) and acetyl-Arg-Arg-chloromethyl ketone (Ac-R-R-CMK) by D.
Russell (Washington University, St. Louis). Cystatin C (human
recombinant) and the mouse monoclonal antibody to cruzain (212 BH6)
were obtained from J. Scharfstein (Universidade Federal do Rio de
Janeiro).Cells and Parasites
Normal rat kidney (NRK)
fibroblasts, Chinese hamster lung (Dede) cells (ATCC 39-CCL), African
green monkey (CV-1) cells (ATCC 70-CCL), and Madin-Darby canine kidney
(MDCK) cells were maintained in 10 mM Hepes-buffered
Dulbecco's modified Eagle's medium, containing 10% FBS. A7
cells, a derivative of a human malignant melanoma cell line M2 (a gift
from C. Cunningham, Brigham and Women's Hospital, Harvard Medical
School, Boston) was maintained in 10 mM Hepes-buffered
Dulbecco's modified Eagle's medium, containing 10% FBS and
500 µg/ml G418. Chinese hamster ovary (CHO) cells were grown in
-minimum Eagle's medium, with 5% FBS. All cells were
maintained at 37 °C in a humidified atmosphere containing 5%
CO
. Trypomastigotes from the T. cruzi Y strain
were obtained from the supernatant of infected monolayers of LLC-MK2
cells as described previously(19) . Epimastigotes, the
noninfective insect stage (Y strain), were cultured in liver infusion
tryptose medium containing 10% FBS at 28 °C(20) .
Trypomastigote invasion of mammalian cells was quantitated as described
previously(11) .Preparation of Parasite Soluble Fraction
Freshly
harvested trypomastigotes or epimastigotes were washed in
Hepes-buffered Ringer's solution containing 0.5% bovine serum
albumin (21) and resuspended at 2 
10
parasites/ml in Dulbecco's phosphate-buffered saline
containing 2 mM Ca and 1 mM Mg (PBS), then killed by
heating at 56 °C for 5 min and frozen at -80 °C.
Freeze-thawed parasites were disrupted by sonication (Model 250,
Branson Ultrasonics, Danbury, CT) on ice with a microtip for two 15-s
bursts at a setting of 2. Unbroken cells and nuclei were removed by
centrifugation at 750 g for 10 min at 4 °C. The
postnuclear supernatant was centrifuged at 100,000 g at 4 °C for 45 min, the resulting supernatant fraction was
passed over a 1-ml column of concanavalin A (ConA)-Sepharose, and the
unbound material was collected, aliquoted, and stored at -80
°C. For experiments in which the soluble glycoprotein fraction was
required, elution from ConA-Sepharose was carried out using a solution
of 0.25 M methyl -D-mannopyranoside, 0.25 M methyl -D-glucopyranoside in PBS.Spectrofluorometric Measurement of Intracellular Free
Calcium in NRK Cells
NRK cells were plated at a density of 1
10
/cm on 9 22-mm coverslips 2
days prior to use. Cells were loaded with 5 µM fura-2/AM,
0.01% pluronic F-127, and 2.5 mM probenecid in Hepes-buffered
Dulbecco's modified Eagle's medium, 5% FBS, for 40 min at
37 °C. Coverslips were rinsed in Ringer's solution and placed
into a cuvette with 1600 µl of Ringer's at 37 °C with
constant stirring in a model F-2000 spectrofluorometer (Hitachi
Instruments, Inc., Danbury, CT). After a 5-min equilibration period,
test solutions were added directly to the cuvette 20-50 s after
beginning to monitor the fluorescence intensity continuously at two
alternating wavelengths (excitation 340 nm, emission 510 nm; excitation
380 nm, emission 510 nm) using software for the measurement of
intracellular cation concentration (Hitachi Instruments, Inc). Each
experiment was run for 500 s and calibrated by adding 10 µM ionomycin at 300 s followed by 100 mM EGTA.
[Ca]
was calculated assuming a K
for fura-2 of 224 nM(22) . For
inhibition experiments, the ConA unbound soluble fraction was incubated
with the following inhibitors for 5 min at 37 °C immediately before
use in the assay: 100 µM leupeptin, aprotinin, E-64,
chymostatin, Z-F-R-FMK, or antipain; 50 µM Ac-R-R-CMK; 300
µM pepstatin A; 40 µg/ml cystatin C; 10 µg/ml
SBTI; or 100 µg/ml heparin. N-Ethylmaleimide (NEM)
treatment was carried out by incubating supernatants on ice with 400
µM NEM for 15 min followed by 400 µM DTT for
15 min. Mock-treated extracts served as controls for all experiments.
In some experiments, the ConA unbound fraction was passed over a 1-ml
column of heparin-agarose equilibrated in PBS.
Unbound material was collected, and, following thorough washing of the
column, bound proteins were eluted with 1.25 M NaCl in
PBS. The heparin-bound fraction was desalted using
Bio-Spin 6 columns (Bio-Rad Laboratories).Assay of Protease Activity
Protease activity was
assayed by following the release of the fluorescent group
7-amino-4-methylcoumarin (AMC) from Z-F-R-AMC. 10 µl of parasite
extract was added to 190 µl of 20 µM substrate in
Tris-buffered saline (TBS; 50 mM Tris-HCl, pH 7.2, 150 mM NaCl) for 15 min at 37 °C, the reactions were stopped by
adding 1 ml of ethanol, and fluorescence was read (excitation 380 nm;
emission 440 nm) as described (18) . To determine the inhibitor
profile of the Z-F-R-AMC hydrolyzing activity, the protease inhibitors
listed above were incubated with 50 µl of the ConA unbound fraction
for 15 min at 22 °C prior to adding aliquots of 10 to 190 µl of
20 µM Z-F-R-AMC in the presence and absence of 5 mM DTT. For direct visualization of protease activity in various
parasite fractions, SDS-PAGE was carried out in 10% acrylamide slab
gels at 4 °C and processed as described previously(18) .
For peptidase detection, washed gels were incubated for 15 min in 20
µM Z-F-R-AMC in TBS, 5 mM DTT, rinsed with
distilled water, and visualized by ultraviolet light (312 nm). Gels
co-polymerized with 0.1% gelatin were electrophoresed at 4 °C,
washed with 2% Triton X-100 at room temperature for 30 min, and
incubated in TBS, 5 mM DTT overnight at 37 °C. Gels were
stained with 0.125% Coomassie Blue, 40% methanol, 10% acetic acid and
destained with 25% methanol, 10% acetic acid to visualize bands of
proteolytic activity(23) .Purification of the 120-kDa Peptidase
The 120-kDa
peptidase was purified following the procedure described (18) with several modifications. Briefly, epimastigotes, grown
and harvested as described above, were resuspended in PBS at a concentration of 5 10/ml, heated to 56
°C for 5 min, then immediately frozen at -80 °C. Parasite
suspensions were thawed, and 20 µM E-64 was added before
preparation of the ConA unbound material (described above). The
flow-through fraction from ConA-Sepharose (50 ml) was applied to a
10-ml column of DEAE-cellulose equilibrated in PBS.
Unbound material containing the 120-kDa enzyme was subjected to
ammonium sulfate precipitation (30-52% saturation) for 2 h on
ice. Precipitated protein was recovered by centrifugation at 15,000
g for 30 min, and pellets were resuspended in
PBS containing 20 µM E-64, filtered
through a 0.22-µm filter unit (Millipore Products Division), and
desalted on a Econo-Pac® 10DG column (Bio-Rad Laboratories)
equilibrated in PBS. 4 ml of desalted material was
applied to Sephacryl 300 (1.6 cm 60 cm) (Pharmacia Biotech
Inc., Uppsala, Sweden) equilibrated in PBS, and
proteins were eluted in the same buffer, collected as 2-ml fractions.
Fractions containing enzyme activity (assayed using Z-F-R-AMC as
described above) were pooled and concentrated by ammonium sulfate
precipitation (70% saturation). The resuspended pellet was desalted
into 25 mM Bis-Tris, pH 6.4, and the material was applied to a
Mono Q HR 5/5 column (Pharmacia Biotech Inc., Uppsala, Sweden). Bound
material was eluted with a gradient of 0-250 mM NaCl, 25
mM Bis-Tris, pH 6.4. The 120-kDa enzyme eluted at a salt
concentration of approximately 75-90 mM NaCl. These
fractions were pooled and applied to a Mono P HR 5/5 chromatofocusing
column (Pharmacia Biotech Inc., Uppsala, Sweden), and bound material
eluted with a pH gradient of 6.4-4.0 formed with Polybuffer 74
(Pharmacia Biotech Inc., Uppsala, Sweden). The enzyme activity eluted
over a pH range of 4.8-5.1 as described previously(18) .
T. cruzi Soluble Fraction Induces Intracellular free
Ca
The
responsiveness of NRK cells was tested in the spectrofluorometric assay
by adding thrombin or bombesin diluted in 500 µl of Ringer's
solution. Additions of 0.2 unit/ml thrombin (Fig. 1A)
or 20 nM bombesin (Fig. 1B) resulted in a
rapid transient increase in the [Ca Transients in Mammalian Cells] in NRK cells. Using similar conditions,
Ca-signaling activity was demonstrated in total
soluble extracts of trypomastigotes, the invasive form of T. cruzi (Fig. 1C), but not in equivalent fractions of
epimastigotes (Fig. 1D). Trypomastigote-induced
[Ca] transients occurred
rapidly, similar to the response seen in these cells after stimulation
with thrombin or bombesin. Fig. 1C represents a typical
response of NRK cells to trypomastigote-soluble material: the response
occurred within 30 s of addition, the peak
[Ca] (400 nM) was
reached in approximately 20 s, and, following the peak, the
[Ca] remained higher than the
basal level for the remainder of the assay. The level of increase of
[Ca] in NRK cells was found to
be proportional to the amount of trypomastigote-soluble fraction added:
dilution of more than 50% resulted in delayed, smaller responses (Fig. 2, A-C). A similar titration effect was
observed when the total soluble material from epimastigotes was used
instead of buffer to dilute the active trypomastigote fraction (Fig. 2, D-F). These results indicate that
epimastigotes do not contain an inhibitor of the trypomastigote
Casignaling factor.
] in NRK cells. 0.2
unit/ml thrombin (A), 20 nM bombesin (B),
total soluble extract (10 parasite eq/ml) of
trypomastigotes (C), or epimastigotes (D) was added
(500 µl in Ringer's solution) to NRK cells loaded with
fura-2/AM. Resulting responses were measured spectrofluorometrically,
and [Ca] was
determined as described under ``Experimental Procedures.'' Arrows indicate the time (s) at which additions were made in
each experiment.
] response in NRK
cells. Trypomastigote soluble extract (A and D) was
diluted 1:1 (B) and 1:4 (C) with Ringer's
solution or diluted 1:1 (E) and 1:4 (F) with the
total soluble extract from epimastigotes (epi) (10 parasite eq/ml). Undiluted or diluted soluble extracts were added
to fura-2-loaded NRK cells in a total volume of 500 µl, and
responses were recorded as described under ``Experimental
Procedures.'' The apparent initial elevation in basal levels of
[Ca] (E and F) is an artifact of calibration caused by the addition of
increasing volumes of epimastigote extract.
parasite eq/ml) were determined, and
the parameters are listed in Table 1. The peak
[Ca], which represents the
maximum increase above the basal
[Ca], was in the range of 340
+ 174 nM (mean + S.D., n = 11). The
latency, which is the time taken to reach a half-maximal response, was
35 + 8 s. Several other cell lines were shown to respond to
soluble trypomastigote material (Table 1), indicating that the
infective stage of T. cruzi is able to induce transient
increases in [Ca] in distinct
cell types derived from several different species (NRK, rat; CHO and
Dede, hamster; MDCK, dog; CV-1, monkey; and A7, human).
The Trypomastigote Ca
Since the parasite Ca-signaling
Activity Involves a Cysteine-like Protease That Is Insensitive to
E-64-signaling activity
was previously shown to be sensitive to proteolysis(15) , the
trypomastigote soluble extract was passed over a ConA-Sepharose column
to remove cruzain, the major cysteine protease of T.
cruzi(24) . Ca-signaling activity was
recovered in the flow-through fraction from the ConA-Sepharose column (Fig. 3A), and no activity was associated with the
glycoprotein fraction eluted with -D-methylmannoside (Fig. 3B). Preliminary efforts to characterize the
trypomastigote factor responsible for eliciting Ca responses in host cells involved further fractionation of the
ConA unbound material in the presence of a mixture of protease
inhibitors. Unexpectedly, we discovered that the addition of protease
inhibitors to the active fraction abolished the response (not shown).
Thus, the effect of individual protease inhibitors on
Ca-signaling activity was subsequently analyzed (Fig. 3, C-H). Individual inhibitors were
incubated with the active (ConA unbound) fraction for 5 min at 37
°C immediately prior to testing for activity. Preincubation with
100 µM leupeptin (Fig. 3C), 100 µM chymostatin (Fig. 3E), 100 µM Z-F-R-FMK (Fig. 3G), 50 µM Ac-R-R-CMK (Fig. 3H), or 100 µM antipain (Table 2), completely impaired the ability of the active fraction
to induce a Ca response. In contrast, 100 µM aprotinin (Fig. 3D), 100 µM E-64 (Fig. 3F), 1 mM phenylmethylsulfonyl fluoride,
300 µM pepstatin A, 100 µg/ml SBTI, or 40 µg/ml
cystatin C (Table 2) failed to inhibit the response. The lack of
inhibition by E-64 was unexpected, since other inhibitors of cysteine
proteases (leupeptin, chymostatin, Z-F-R-FMK, Ac-R-R-CMK, and antipain)
were very effective in blocking the Ca signal.
] transients in NRK
cells by protease inhibitors. Trypomastigote total soluble extract (500
µl) retained activity after passage through a ConA-Sepharose column (A). No activity was eluted in the glycoprotein fraction with
0.25 M methyl -D-mannopyranoside, 0.25 M methyl -D-glucopyranoside (B). ConA unbound
fraction (500 µl) was incubated for 5 min at 37 °C with 100
µM leupeptin (C), 100 µM aprotinin (D), 100 µM chymostatin (E), 100
µM E-64 (F), 100 µM Z-F-R-FMK (G) or 50 µM Ac-R-R-CMK (H) before
adding to NRK cells in the spectrofluorometric assay at the times
indicated by arrows.
response elicited by thrombin (Table 3),
indicating that added inhibitors did not interfere unspecifically with
the fluorescence assay. The peak [Ca] was consistently greater when thrombin was added to NRK cells in
the presence of leupeptin or chymostatin. The reasons for this
observation are unknown, but it is possible that the target of these
inhibitors were thrombin degrading enzymes analogous to cell surface
peptidases described to degrade peptide agonists in other
systems(25) .
Inhibitors of the Ca
The results described above
indicated that a protease with a specific inhibitor profile was
involved in the generation of [Ca-signaling Activity
Inhibit Z-F-R-AMC Hydrolysis] transients by T. cruzi extracts. The two possible
sources of the protease were the NRK cells used as targets in the assay
or the trypomastigote ConA unbound fraction. Since the peptide
Z-F-R-FMK was an effective inhibitor of the
Ca-response (Fig. 3G), the same
peptide conjugated to a cleavable fluorescent group (AMC) was used to
detect hydrolytic activity in both NRK cells and in the trypomastigote
active soluble fraction. Enzyme assays using Z-F-R-AMC as a substrate
were carried out in Ringer's solution (pH 7.2) at 37 °C
(since these were the conditions for the
[Ca] assays) or in 50 mM Tris-HCl, pH 7.2, 150 mM NaCl with essentially the same
results. signal induced in these cells by trypomastigote
extracts (not shown). Therefore, the presence of Z-F-R-AMC hydrolyzing
activity in the trypomastigote ConA unbound fraction was investigated.
The protease inhibitors listed in Table 2(some of which appear
in Fig. 3) were tested at the same final concentrations
previously found to inhibit the Ca-signaling activity
of trypomastigote fractions. As shown in Table 2, agents that had
a strong inhibitory effect on the Ca-signaling
activity (leupeptin, chymostatin, Z-F-R-FMK, antipain, and Ac-R-R-CMK)
also inhibited a peptidase activity present in these fractions.
Similarly, aprotinin, phenylmethylsulfonyl fluoride, E-64, SBTI, and
pepstatin A failed to inhibit either the Ca-signaling
activity or peptidase activity. Cystatin C, an inhibitor of papain-like
cysteine proteases(26) , had no effect on either the
Ca response or the peptidase activity (Table 2). In addition, 10 mM EDTA and 100 µM 1-10-phenanthroline had no effect on Z-F-R-AMC hydrolysis by
trypomastigote extracts, while 5 µM HgCl totally abolished it (not shown). As also shown in Table 2,
the Z-F-R-AMC hydrolyzing activity was only slightly increased under
reducing conditions.A 120-kDa Z-F-R-AMC Hydrolyzing Enzyme Is Present in
Fractions with Ca
Two
methods to directly visualize proteases in SDS-PAGE gels were used to
analyze soluble fractions of T. cruzi: hydrolysis of
co-polymerized gelatin or of Z-F-R-AMC added as a substrate after
electrophoresis. In gelatin gels, several bands of proteolytic activity
were observed in the trypomastigote total soluble extract (Fig. 4A, Trypo T). The major protease bands
which migrated at approximately 40-50 kDa were shown to
correspond to cruzain, the well-characterized major cysteine protease
of T. cruzi(24) , by Western blot with the monoclonal
antibody 212 BH6 (27; not shown). These bands as well as others with
proteolytic activity were removed by binding to ConA-Sepharose (Fig. 4A, Trypo UB) and recovered by elution
of the column with specific sugars (Fig. 4A, Trypo
GP). Using Z-F-R-AMC as a substrate, the only detectable activity
in the ConA unbound fraction was a band migrating at approximately 120
kDa (Fig. 4B, Trypo UB), not detected in
gelatin gels (Fig. 4A, Trypo UB). This band
was also present in the trypomastigote total soluble extract (Fig. 4B, Trypo T) but was absent in the
ConA-binding fraction (Fig. 4B, Trypo GP). We
have been unable to detect additional hydrolytic activities in the ConA
unbound fraction using macromolecular substrates such as gelatin and
casein or the peptide substrates Z-F-R-AMC and Z-G-G-R-AMC (not shown).
The ConA unbound fraction prepared from epimastigote forms of T.
cruzi contained the 120-kDa Z-F-R-AMC hydrolyzing activity (Fig. 4B, Epi UB), and this activity was
absent from the epimastigote ConA-binding fraction (Fig. 4B, Epi GP). As demonstrated previously (15) and above (Fig. 1D), epimastigotes are not
able to induce Ca-signaling Activity responses in host cells.
10
parasite eq/lane, and electrophoresis
was carried out under nonreducing conditions at 4 °C (18) .
Before visualization of proteolytic activity, gels were washed in TBS,
2% Triton X-100 as described under ``Experimental
Procedures.'' A, gel (containing 0.1% gelatin) was
incubated overnight at 37 °C in TBS, 5 mM DTT, then
stained with Coomassie Blue, and destained to visualize regions of
gelatin degradation(23) . Arrows point to bands
corresponding to cruzain, the T. cruzi major cysteine
protease(24) . B, gel was incubated at 37 °C in 20
µM Z-F-R-AMC in TBS for 15 min(18) . Fluorescent
bands which appear in the region of substrate hydrolysis were
visualized on a UV light box (312 nm). C, trypomastigote ConA
unbound fraction (10
parasite eq) was run under nonreducing
conditions at 4 °C. Vertical strips of gel (1 cm in width) were
incubated for 10 min at 22 °C in TBS alone (lane 1) or TBS
containing 100 µM leupeptin (lane 2), 100
µM E-64 (lane 3), or 100 µM Z-F-R-FMK (lane 4), before addition of 20 µM Z-F-R-AMC and incubation as in B. Arrows in B and C point to the 120-kDa
peptidase.
activity, the ConA unbound
fraction of trypomastigotes was separated on preparative SDS-PAGE, and
the gel was cut in strips and treated with different protease
inhibitors, before incubation with Z-F-R-AMC. Leupeptin (Fig. 4C, lane 2) and Z-F-R-FMK (Fig. 4C, lane 4) completely abolished
hydrolysis of Z-F-R-AMC by the 120-kDa peptidase, while E-64 had no
effect (Fig. 4C, lane 3). These results, taken
together with the observations described in the previous section,
indicate that the 120-kDa enzyme detected in fractions with
Ca-signaling activity is identical with a T.
cruzi alkaline peptidase previously purified from epimastigote
stages of the parasite(18) .A Trypomastigote-specific Factor Is Also
Required
Several observations indicate that the 120-kDa
peptidase alone is not sufficient to generate
[Ca] transients in mammalian
cells. First, the 120-kDa peptidase was detected in epimastigote
soluble extracts at a similar specific activity to that detected in
equivalent fractions of trypomastigotes (Fig. 4B and Fig. 5A), but the epimastigote extracts were not able
to induce [Ca] transients in
mammalian cells ( Fig. 1and Fig. 5). It is unlikely that
epimastigotes express an inhibitor of the
Ca-signaling activity since dilution of the
trypomastigote active fraction with epimastigote soluble extract was
not inhibitory (Fig. 2, D-F). Secondly, a
fraction highly enriched in the 120-kDa enzyme (280-fold purification),
from a Mono P chromatofocusing column which contained 6 bands in
addition to the peptidase (Fig. 5B), did not induce any
increase in [Ca] when added to
NRK cells (Fig. 5A).
-signaling activities associated with
trypomastigotes, epimastigotes and partially purified 120-kDa
peptidase. A, soluble extracts from trypomastigotes and
epimastigotes and partially purified 120-kDa peptidase were prepared
and assayed for Z-F-R-AMC hydrolysis or Ca-signaling
activity as described under ``Experimental Procedures.'' Fl. mg
min represents
fluorescence units of liberated AMC per mg of protein per min. B, silver stain profile of the Mono P fraction that is highly
enriched in 120-kDa peptidase (280-fold purification). The arrow indicates the position corresponding to a fluorescent band
visualized in the same sample after incubation with Z-F-R-AMC (not
shown). Ca-signaling activity is present only in
trypomastigote soluble fractions, not in epimastigote fractions
containing a similar amount of the 120-kDa peptidase. Partially
purified 120-kDa peptidase lacks Ca-signaling
activity.
-signaling activity. These were the
interaction with heparin and alkylation with N-ethylmaleimide
(NEM). Trypomastigote Ca-signaling activity was lost
when the active extract (Fig. 6A) was passed over a
heparin-agarose column (Fig. 6B), whereas the 120-kDa
peptidase was recovered in the flow-through fraction (Fig. 6F, after heparin). Addition of soluble heparin
(100 µg/ml) to the trypomastigote extract resulted in a partial
inhibition of Ca-signaling activity (Fig. 6C) while having no inhibitory effect on the
120-kDa peptidase (Fig. 6F, soluble heparin). To
control for possible inhibition of the Ca response
due to heparin leakage from the agarose column, buffer was passed
through the column under identical conditions and mixed with fresh
trypomastigote extract. This was found not to be inhibitory (not
shown). The heparin-binding fraction eluted from the column was not
able to induce a Ca response (not shown) and
contained no Z-F-R-AMC hydrolytic activity (Fig. 6F, Heparin-bound). N-Ethylmaleimide pretreatment (400
µM) of the trypomastigote extract followed by quenching
unreacted NEM with 400 µM DTT completely inhibited the
response (Fig. 6D), whereas pretreatment with DTT (400
µM) alone had no effect (Fig. 6E). Again,
activity of the 120 kDa peptidase was not inhibited by NEM/DTT
treatment or DTT alone (Fig. 6F). These data indicate
that the activity of the 120-kDa peptidase alone is insufficient for
inducing Ca responses in host cells, and that an
NEM-sensitive heparin-binding component(s) from trypomastigotes is also
required. Attempts to reconstitute Ca-signaling
activity by combining the heparin-bound and unbound fractions have not
been successful.
-signaling but not peptidase activity. Soluble
trypomastigote extract contained both Ca-signaling
activity (A) and peptidase activity as measured by hydrolysis
of Z-F-R-AMC (F, before heparin). After passing extract
through a heparin-agarose column, Ca-signaling
activity was lost (B), while peptidase activity was recovered (F, after heparin). Addition to the soluble extract of 100
µg/ml heparin (C) or 400 µM NEM followed by
400 µM DTT (D) as described under
``Experimental Procedures'' resulted in the inhibition of
Ca-signaling, while not affecting the peptidase
activity (F, soluble heparin; NEM/DTT). DTT alone did
not affect either Ca-signaling (E) or
peptidase activity (F, DTT).
Membrane-permeable Inhibitors of the 120-kDa Peptidase
Block Invasion of NRK Cells by T. cruzi
Previous studies had
shown that inhibition of the [Ca] transients induced in NRK cells by trypomastigotes blocked
invasion(15) . Therefore, we proceeded to verify if inhibitors
of the 120-kDa peptidase had the same effect. In order to ensure
penetration of the inhibitors into parasite intracellular compartments,
we used small peptide inhibitors shown previously to be
membrane-permeable(28, 29) . Trypomastigotes were
incubated with the inhibitors at different final concentrations for 15
min at 37 °C in Ringer's solution prior to adding to NRK
cells for invasion. Under these conditions, Z-F-R-FMK and Ac-R-R-CMK
had a significant inhibitory effect on invasion levels, in a
dose-dependent manner (Fig. 7).
agonist activity for mammalian cells.
Previous studies performed at the single cell level showed that the
invasive form of T. cruzi induces repetitive cytosolic free
Ca transients in NRK cells, and prevention of these
transients interferes with the invasion process(15) . In order
to characterize the Ca-signaling activity in
trypanosomes, a spectrofluorometric method was employed to
quantitatively measure [Ca] in
populations of host cells in response to parasite extracts. We have
found that substantial increases in
[Ca] were induced in many cell
types by trypomastigote extracts and that these transients were
comparable to those induced by the soluble agonists thrombin and
bombesin. In keeping with previous results(15) , extracts of
epimastigotes, the noninfective stage of T. cruzi, failed to
induce a Ca response in NRK cells.-signaling activity was found to
be soluble after disruption of parasites. Although some activity was
associated with the crude membrane fraction as previously
observed(15) , it could be removed by repeated washing (not
shown), thus leading to the conclusion that the
Ca-signaling activity of trypomastigotes is not
tightly associated with membranes.-signaling activity in the trypomastigote soluble
fraction. Pretreatment of the active fraction with a specific subset of
protease inhibitors (leupeptin, chymostatin, Z-F-R-FMK, Ac-R-R-CMK, or
antipain) resulted in the inhibition of the Ca response in NRK cells, whereas other inhibitors (aprotinin, SBTI,
phenylmethylsulfonyl fluoride, pepstatin A, and E-64) had no effect ( Fig. 3and Table 2). Although several proteases were
detected in the crude soluble fraction by hydrolysis of gelatin in
SDS-PAGE gels, most were removed by binding to ConA-Sepharose (Fig. 4A), including a diffuse band migrating between
40 and 60 kDa which corresponds to the well-characterized major T.
cruzi cysteine protease, cruzain(24) . Additional
ConA-binding proteins probably correspond to other previously detected T. cruzi proteases(30) . -signaling activity persisted after removal of
proteases by ConA-Sepharose; therefore, additional hydrolytic
activities in the ConA unbound fraction were sought. Since Z-F-R-FMK, a
fluoromethyl ketone-derivatized peptide which irreversibly inhibits
cysteine proteases (31) effectively blocked the
Ca-signaling activity, the same peptide coupled to a
cleavable fluorescent group (AMC) was used as a substrate to detect
hydrolytic activity in the active fraction. Hydrolysis of Z-F-R-AMC by
an enzyme present in the active fraction was found to be sensitive to
the same subset of protease inhibitors that were effective in blocking
the Ca signal (Table 2). Using Z-F-R-AMC as a
substrate, the only detectable proteolytic activity in the ConA unbound
fraction migrated at an approximate molecular mass of 120 kDa on
SDS-PAGE gels (Fig. 4B). In similar gels, the inhibitor
profile of the 120-kDa enzyme was shown to be identical with that of
the Ca-signaling activity. Therefore, based on the
excellent correlation between inhibitors of the
Ca-signaling activity and inhibitors of the 120-kDa
enzyme, there is good evidence to support the involvement of the
120-kDa protease in
[Ca]-signaling.] transients in host cells.
First, the noninvasive forms of the parasite, epimastigotes, express an
active enzyme with identical biochemical characteristics (16, 18; Fig. 4B, Epi UB), yet this parasite life cycle
stage is incapable of inducing a [Ca] response in mammalian cells. Secondly, when the trypomastigote
active fraction was passed over heparin-agarose,
[Ca]-signaling activity was
lost. The 120-kDa enzyme does not bind to heparin, nor is it sensitive
to soluble heparin, whereas
[Ca]-signaling activity was
shown to be heparin-sensitive. Similarly, N-ethylmaleimide
pretreatment of the trypomastigote active fraction abolished
[Ca]-signaling activity but did
not affect the activity of the 120-kDa enzyme. These results suggest
that, in addition to the 120-kDa alkaline peptidase, an NEM-sensitive,
heparin-binding component(s) is required for the generation of the
[Ca] signal. The localization
of the 120-kDa enzyme and its putative substrate are not known. Since
solubilization of Ca-signaling activity occurs
readily in the absence of detergent, it may be located in an
intracellular compartment and possibly secreted from the parasite upon
contact with the host cell.-signaling
activity, are potent inhibitors of host cell invasion by T.
cruzi. We have ruled out the participation of the major cysteine
protease in Ca-signaling for two reasons: removal of
cruzain (and other ConA-binding proteases) from the soluble fraction
did not impair Ca-signaling activity and E-64, which
inhibits cruzain at low concentrations(33) , failed to inhibit
activity.] transients in host cells.
The activity of this factor is short-lived, since no
Ca-signaling activity persists when the 120-kDa
peptidase is blocked by inhibitors in a 5-min preincubation. This
observation provides an additional argument for the tight coupling of
the 120-kDa peptidase with Ca-signaling activity and
explains our difficulty in isolating the active signaling molecule from
trypomastigote soluble extracts.-signaling
factor produced by T. cruzi has properties of a mammalian
hormone or growth factor, in the sense that it is proteolytically
generated and responses occur a few seconds after contact with target
cells. In addition, recent results from our laboratory show that the
trypomastigote soluble fraction induces rapid polyphosphoinositide
hydrolysis with inositol triphosphate production and subsequent
Ca mobilization from intracellular stores in NRK
cells. ()This is a response characteristic of the binding of
hormones and growth factors to their specific receptors(36) . ],
and interference with the [Ca] transients inhibits invasion. Since invasion involves recruitment
and fusion of host cell lysosomes(11) , it is possible that the
signaling factor described here plays a role in this process. Several
trypomastigote surface molecules have also been implicated in the T. cruzi invasion
mechanism(37, 38, 39, 40, 41, 42) ,
while host cell sialic acid moieties and heparan sulfate
glycoconjugates seem to play a role as targets for parasite binding (43, 44) . The precise mechanism of T. cruzi entry into mammalian cells remains to be fully elucidated.-signaling factor for
mammalian cells. The downstream effects of this signaling event are not
yet known, but may be part of a communication process which regulates
early events in host cell invasion by this
parasite(11, 15) .
))
We thank J. Scharfstein for kindly providing cystatin
C, monoclonal antibodies to cruzain, and many helpful discussions. We
are also grateful to J. McKerrow and D. Russell for their gifts of
protease inhibitors, to P. Novick and members of his laboratory for
fast protein liquid chromatography use, and J. Bouvier for advice.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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