©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A 120-kDa Alkaline Peptidase from Trypanosoma cruzi Is Involved in the Generation of a Novel Ca-signaling Factor for Mammalian Cells (*)

(Received for publication, November 17, 1994; and in revised form, January 4, 1995)

Barbara A. Burleigh (§) Norma W. Andrews

From the Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Trypomastigotes, the infective stages of the intracellular parasite Trypanosoma cruzi, induce rapid and repetitive cytosolic free Ca 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.


INTRODUCTION

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]) 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) .

The present study was undertaken to characterize the trypomastigote factor mediating Ca-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.


EXPERIMENTAL PROCEDURES

Materials

Reagents were obtained from the following sources: fura-2/AM and pluronic F-127 were obtained from Molecular Probes, Inc. Methyl alpha-D-mannopyranoside, methyl alpha-D-glucopyranoside, probenecid, EGTA, leupeptin, aprotinin, chymostatin, soybean trypsin inhibitor (SBTI), (^1)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 alpha-minimum Eagle's medium, with 5% FBS. All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO(2). 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 times 10^8 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 times g for 10 min at 4 °C. The postnuclear supernatant was centrifuged at 100,000 times 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 alpha-D-mannopyranoside, 0.25 M methyl alpha-D-glucopyranoside in PBS.

Spectrofluorometric Measurement of Intracellular Free Calcium in NRK Cells

NRK cells were plated at a density of 1 times 10^4/cm^2 on 9 times 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](i) was calculated assuming a K(d) 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 times 10^8/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 times 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 times 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) .


RESULTS

T. cruzi Soluble Fraction Induces Intracellular free Ca Transients in Mammalian Cells

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](i) 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](i) 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](i) (400 nM) was reached in approximately 20 s, and, following the peak, the [Ca](i) remained higher than the basal level for the remainder of the assay. The level of increase of [Ca](i) 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.


Figure 1: Total soluble extract of T. cruzi trypomastigotes induces elevation in [Ca] in NRK cells. 0.2 unit/ml thrombin (A), 20 nM bombesin (B), total soluble extract (10^8 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.




Figure 2: Titration of [Ca] 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) (10^8 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.



The characteristics of an average response of NRK cells to 500 µl of trypomastigote total soluble fraction (10^8 parasite eq/ml) were determined, and the parameters are listed in Table 1. The peak Delta[Ca](i), which represents the maximum increase above the basal [Ca](i), 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](i) 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-signaling Activity Involves a Cysteine-like Protease That Is Insensitive to E-64

Since the parasite Ca-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 alpha-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.


Figure 3: Inhibition of parasite-induced [Ca] 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 alpha-D-mannopyranoside, 0.25 M methyl alpha-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.





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 response elicited by thrombin (Table 3), indicating that added inhibitors did not interfere unspecifically with the fluorescence assay. The peak [Ca](i) 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-signaling Activity Inhibit Z-F-R-AMC Hydrolysis

The results described above indicated that a protease with a specific inhibitor profile was involved in the generation of [Ca](i) 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](i) assays) or in 50 mM Tris-HCl, pH 7.2, 150 mM NaCl with essentially the same results.

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 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(2) 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-signaling Activity

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 responses in host cells.


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 times 10^7 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 (10^9 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.



To demonstrate that the 120-kDa peptidase exhibited the same sensitivity to inhibitors of the Ca 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](i) 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](i) 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](i) when added to NRK cells (Fig. 5A).


Figure 5: Comparison of peptidase and Ca-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. mgmin 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.



Two additional conditions were found in which the presence of an active 120-kDa enzyme did not correlate with Ca-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.


Figure 6: Interaction with heparin and NEM inhibits Ca-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](i) 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).


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) .




DISCUSSION

We have provided, for the first time, evidence that an intracellular parasite employs proteolytic processing to generate a factor with Ca 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](i) in populations of host cells in response to parasite extracts. We have found that substantial increases in [Ca](i) 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.

The majority of the trypomastigote Ca-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.

Our results indicate that proteolytic activity is tightly coupled to Ca-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) .

Ca-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](i)-signaling.

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](i) 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](i) response in mammalian cells. Secondly, when the trypomastigote active fraction was passed over heparin-agarose, [Ca](i)-signaling activity was lost. The 120-kDa enzyme does not bind to heparin, nor is it sensitive to soluble heparin, whereas [Ca](i)-signaling activity was shown to be heparin-sensitive. Similarly, N-ethylmaleimide pretreatment of the trypomastigote active fraction abolished [Ca](i)-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](i) 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.

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-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.

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](i) 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.

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-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. (^2)This is a response characteristic of the binding of hormones and growth factors to their specific receptors(36) .

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](i), and interference with the [Ca](i) 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.

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-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) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grants RO1AI32056 and RO1AI34867 (to N. W. A.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a training award from the Medical Research Council of Canada.

(^1)
The abbreviations used are: SBTI, soybean trypsin inhibitor; DTT, dithiothreitol; NEM, N-ethylmaleimide; AMC, 7-amino-4-methylcoumarin; FMK, fluoromethyl ketone; CMK, chloromethyl ketone; FBS, fetal bovine serum; PBS, phosphate-buffered saline; ConA, concanavalin A; PAGE, polyacrylamide gel electrophoresis; Bis-Tris, 2-[(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.

(^2)
A. Rodriguez, M. Rioult, A. Ora, and N. W. Andrews, submitted for publication.


ACKNOWLEDGEMENTS

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.


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