Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002
An early event in the Trypanosoma cruzi cell invasion process, the recruitment of host lysosomes, led us to investigate the involvement of signal transduction. Infective trypomastigotes were found to contain a soluble Ca2+-signaling activity for mammalian cells that is sensitive to protease inhibitors. Inhibitor and substrate utilization profiles were used to purify a candidate peptidase for involvement in this process, from which we isolated a full-length cDNA clone. The sequence revealed a novel enzyme, denominated T. cruzi oligopeptidase B, which is homologous to members of the prolyl oligopeptidase family of serine hydrolases, known to participate in the maturation of biologically active peptides. The T. cruzi oligopeptidase B was expressed as a fully active product in Escherichia coli, and antibodies to the recombinant enzyme inhibited both peptidase activity and Ca2+ signaling induced in normal rat kidney cells by trypomastigote extracts. Our data suggest that the T. cruzi oligopeptidase B participates in processing events in the cytoplasm of the parasites, generating a factor with Ca2+-signaling activity for mammalian cells.
Signal transduction events induced in host cells by intracellular pathogens are emerging as important
regulators of the invasion process (Bliska et al.,
1993 Recent findings from several laboratories indicate that
the cell invasion mechanism of the protozoan parasite Trypanosoma cruzi involves receptor-mediated signal transduction (Ming et al., 1995 Consistent with their infective capabilities, metacyclics
(Dorta et al., 1995 Further characterization of the soluble trypomastigote
Ca2+-signaling activity revealed that the induction of Ca2+
transients in mammalian cells is coupled to the activity of a parasite peptidase (Burleigh and Andrews, 1995 The goal of the present study was to clarify these issues,
by characterizing the peptidase at the molecular level and
by determining its intracellular location and requirement
for Ca2+ signaling. Cloning and nucleotide sequencing of a
full-length cDNA clone revealed that the T. cruzi peptidase is a cytosolic enzyme closely related to members of
the prolyl oligopeptidase family of serine endopeptidases,
some of which were previously shown to function in eukaryote prohormone processing (Fuller et al., 1988 Cells and Parasites
Normal rat kidney (NRK) fibroblasts were maintained in 10 mM Hepesbuffered DME containing 10% FBS at 37°C in a humidified atmosphere
containing 5% CO2. Trypomastigotes from the T. cruzi Y strain were obtained from supernatants of infected monolayers of LLC-MK2 cells as previously described (Andrews et al., 1987 Preparation of Parasite Soluble Extract
Freshly harvested T. cruzi were washed in Hepes-buffered Ringer's solution containing 0.5% BSA (Heuser, 1989 Peptidase Purification and Amino Acid Sequencing
Purification of the 120-kD T. cruzi peptidase was carried out as previously
described (Burleigh and Andrews, 1995 Isolation of a Peptidase cDNA Clone
Total cellular RNA was isolated from T. cruzi using a guanidinium-thiocyanate extraction method (Chomczynski and Sacchi, 1987 Expression of Recombinant Peptidase
in Escherichia coli
The peptidase gene was amplified from the full-length cDNA clone in
pBluescript using the LA-PCRTM kit (Takara Biomedicals, Otsu, Shiga,
Japan) using forward (5 Generation and Affinity Purification
of Anti-Peptidase Antibodies
All antibodies to the T. cruzi oligopeptidase B were raised by the Pocono
Rabbit Farm & Laboratory, Inc. (Canadensis, PA). Antibodies to the denatured 120-kD peptidase were obtained by immunizing a guinea pig with
polyacrylamide gel slices containing 5 µg of protein for the initial immunization and 2 µg for subsequent boosts. Serum was collected biweekly and
analyzed for peptidase-specific antibodies on Western blots of trypomastigote soluble fractions. Neutralizing antibodies were raised by immunizing a rabbit with 200 µg purified rPEP in complete Freund's adjuvant, followed by monthly boosts of 50 µg in incomplete Freund's adjuvant. Silver stain of an overloaded SDS-PAGE gel confirmed the absence of contaminants in the rPEP preparation obtained by nickel affinity chromatography (not shown). IgG from antipeptidase sera was purified using protein A-Sepharose (Pharmacia) according to a standard procedure (Harlow and Lane, 1988 SDS-PAGE and Immunoblotting
Parasite soluble fractions or rPEP were treated with Laemmli sample
buffer (Laemmli, 1970 Peptidase Activity Assays
Peptidase activity was carried out either as a soluble assay using 7-amino4-methyl coumarin (AMC) or 7-amino-4-trifluoromethyl coumarin (AFC)-
conjugated peptide substrates (Enzyme Systems Products), or by direct
detection of peptide-AMC hydrolysis in SDS-PAGE gels as described
previously (Burleigh and Andrews, 1995 Detection of Expressed Products of the Peptidase Gene
in T. cruzi
First strand cDNA was synthesized from total RNA isolated from different T. cruzi life cycle stages as described above for trypomastigotes. 1 µl
of first strand cDNA synthesis reactions from the three life cycle stages
(trypomastigotes, amastigotes, and epimastigotes) was used as template
for PCR with a primer pair (forward primer: 5 Genomic DNA Isolation and Southern Blots
Genomic DNA was isolated from epimastigotes (Y strain) following
published methods (Medina-Acosta and Cross, 1993 Inhibition of Peptidase and Ca2+-Signaling Activities
with Anti-recombinant Peptidase Antibodies
Inhibition of oligopeptidase B activity in trypomastigote soluble extract
(TSE) with IgG from rabbits immunized with the purified recombinant
enzyme was carried out in solution by adding 0.75 µg/ml to 250 µg/ml purified IgG to 50 µl TSE in a final volume of 100 µl. After a 60-min incubation at 4°C, oligopeptidase B activity was measured by adding 200 µl TBS
containing 20 µM Z-Phe-Arg-AMC and 10 µM E-64, and incubated for 10 min at 37°C. The reactions were stopped by adding 1 ml ethanol and AMC
release was measured as described above. To test for the effect of antirPEP antibodies on the soluble Ca2+-signaling activity in these samples,
TSE was preincubated with 200 µg/ml preimmune IgG or anti-rPEP IgG
for 60 min at 4°C, and then added to NRK fibroblasts preloaded with 5 µM
fura-2/AM, 0.01% pluronic F-127 (Molecular Probes Inc., Eugene OR),
and 2.5 mM probenecid (Sigma Chemical Co.). Changes in intracellular
calcium concentration of NRK cells were continuously measured in a
spectrofluorimeter equipped with intracellular cation determination software as described previously (Burleigh and Andrews, 1995 EM Immunolocalization
Trypomastigotes freshly harvested from LLC-MK2 tissue-culture supernatants (1 × 107 per ml) were fixed with an equal volume of 2× fixative
(0.2% glutaraldehyde, 8% formaldehyde, 200 mM Hepes, pH 7.4) and
centrifuged for 10 min at 3,000 g. The supernatant was removed, and fresh 1× fixative was added to the pellet for an additional 2 h. Pellets were infiltrated with 2.1 M sucrose/PBS and frozen in liquid nitrogen. Cryosectioning and labeling of parasites was carried out as described previously
(Webster and Grab, 1988 Isolation of a Full-Length cDNA Clone Encoding the
T. cruzi Peptidase Implicated in Ca2+ Signaling
The T. cruzi peptidase was purified several hundredfold
from epimastigote soluble extracts in several FPLC steps
as described previously (Burleigh and Andrews, 1995
Recombinant T. cruzi Oligopeptidase B
Expressed in E. coli Has Identical Biochemical
Properties to the Native Enzyme
The peptidase cDNA sequence (Fig. 1), which predicts a
polypeptide of 714 amino acids with a calculated molecular mass of 81,011 daltons, was cloned into pET-19b and
expressed in E. coli. Preliminary characterization of the
purified polyhistidine-tagged rPEP revealed that the specific activity of the enzyme, using Z-Phe-Arg-AMC as a
substrate, was similar to that described for the native peptidase purified from epimastigotes (Burleigh and Andrews,
1995
The T. cruzi oligopeptidase B eluted from S-300 gel filtration columns in fractions where proteins of ~180 kD
would be expected (Fig. 2 B). The only detectable immunoreactivity on a Western blot probed with anti-peptidase
antibodies was associated with the pooled peak of peptidase activity in fractions 64-76 (Fig. 2 B). No immunoreactivity was detected in the pool containing fractions 77-89,
which would contain monomeric proteins in the 60-100-kD
range, suggesting that the peptidase is not present in monomeric form in parasite extracts. A similar profile was observed for elution of the purified recombinant oligopeptidase B from an S-300 column (not shown).
Previous studies of the native T. cruzi oligopeptidase B
revealed that this enzyme is specific for peptide substrates
with basic amino acids residues in the P1 position (Ashall,
1990 The results presented above demonstrate that the biochemical properties of the recombinant oligopeptidase B
are identical to those of the peptidase purified from T. cruzi
(Burleigh and Andrews, 1995 T. cruzi Oligopeptidase B Is Homologous to Members of
the Prolyl Oligopeptidase Family of Serine Hydrolases
Comparison of the peptidase cDNA sequence with available databases revealed that the T. cruzi oligopeptidase B
shares significant homology with a subgroup of serine hydrolases referred to as the prolyl oligopeptidase family.
The GxSxGGzz consensus sequence (where x = any
amino acid and z = a hydrophobic residue; Barrett and
Rawlings, 1992
Oligopeptidase B Gene is Present as a Single Copy and
Expressed in All Life Cycle Stages of T. cruzi
Southern blot analysis of T. cruzi genomic DNA probed
with the full-length oligopeptidase B cDNA revealed a
simple pattern after single enzyme digestions. A single
band was produced with several different enzymes, and
two bands were obtained after digestion with SacI, ScaI, or
PvuI (Fig. 4 A). These results are consistent with the predicted restriction map of the peptidase cDNA (not shown),
suggesting that the peptidase gene is represented as a single copy in the T. cruzi Y strain genome.
From previous work it was known that oligopeptidase B
is expressed in trypomastigotes and epimastigotes (Burleigh and Andrews, 1995 Antibodies to the Recombinant T. cruzi Oligopeptidase
B Inhibit Peptide Hydrolysis and Ca2+-Signaling
Activity of Trypomastigote Soluble Extracts
Polyclonal antibodies raised against gel-purified 120-kD
peptidase were unable to inhibit the activity of the enzyme
in solution or to precipitate native enzyme in the absence
of detergent (data not shown). Therefore, highly purified
active rPEP was used to immunize rabbits, and IgG prepared from immune sera was tested for peptidase-blocking
activity. As shown in Fig. 5 A, anti-rPEP IgG proved capable of inhibiting activity of oligopeptidase B in TSE,
whereas preimmune rabbit IgG added at the same final
concentrations had no effect. The maximum inhibitory
effect (90%) achieved with 100 µg/ml anti-rPEP IgG (Fig.
5 A) was not increased with higher concentrations of IgG
(up to 500 µg/ml) or by extending the preincubation time
(not shown). On a Western blot of total soluble trypomastigote proteins separated by SDS-PAGE under reducing
conditions, anti-rPEP IgG specifically recognized the 80-kD
peptidase (Fig. 5 B,
To test whether the inhibition of oligopeptidase B activity by anti-rPEP would affect the Ca2+-signaling activity of
trypomastigote extracts (Tardieux et al., 1994 To directly visualize the kinetics and intensity of the
Ca2+ transients triggered in NRK cells by trypomastigote
soluble extracts, fluorescence video microscopy at the single
cell level was performed after pretreatment with peptidase-blocking antibodies or preimmune IgG (Fig. 6). Similar to the spectrofluorimetric data, pretreatment of TSE
with anti-rPEP antibodies (Fig. 6, panels I) resulted in a
delayed response, and fewer cells responded in comparison with cells preincubated with preimmune IgG (Fig. 6,
panels PI). The time required for the onset of a Ca2+ transient after addition of control or immune IgG-treated TSE was determined in eight separate experiments (Table I).
NRK cells receiving anti-rPEP IgG-treated TSE consistently showed a delayed response when compared with
cells receiving preimmune treated TSE. Therefore, this data
demonstrate a direct link between the activity of T. cruzi
oligopeptidase B and the generation of Ca2+ transients
in mammalian cells. As observed with the native purified T. cruzi oligopeptidase B (Tardieux et al., 1994
Table I.
Effect of Anti-rPEP IgG on the Time of Onset
of [Ca2+]i Elevation Triggered in NRK Cells
by Trypomastigote Soluble Extracts
). As opposed to intracellularly targeted toxins, which
have been extensively studied, very little is known about
pathogen products that trigger signaling pathways through mammalian surface receptors. Experimental evidence suggests that factors capable of modulating the behavior of
mammalian cells are produced by certain intracellular bacteria (Galan, 1994
; Menard et al., 1996
; Yamamoto et al.,
1996
), but their molecular nature and mechanism of action
are largely unknown. In the case of Shigella, an isolated
complex containing the gene products IpaB and IpaC was
shown to induce membrane ruffling and macropinocytosis
in epithelial cells, similar to what is observed during bacterial internalization (Menard et al., 1996
). Although the
mode of action and cellular target of the Ipa complex have
not yet been determined, it was proposed that these secreted proteins may bind to a surface receptor (Menard et
al., 1996
).
; Rodriguez et al., 1995
; Barr et
al., 1996
). Entry of T. cruzi into nonphagocytic mammalian cells occurs by recruitment and fusion of host lysosomes at the parasite attachment site, an unusual process
that results in formation of a parasitophorous vacuole with
lysosomal properties (Tardieux et al., 1992
; Rodriguez et al.,
1996
). The parasite resides in this lysosome-derived vacuole for a short period after invasion, after which it escapes
into the cytoplasm, where replication occurs (Meirelles
and de Souza, 1983; Ley et al., 1990
). T. cruzi invasion of
nonphagocytic mammalian cells is restricted to two life cycle stages: metacyclic forms that are transmitted by the insect vector, and trypomastigotes that are released from infected host cells. Epimastigotes are noninfective forms
that replicate in the insect vector, and amastigotes are the
intracellular stages that replicate inside host cells. Although T. cruzi exhibits tropism for specific cell types in the vertebrate host, it is able to infect many different cell types in culture (Brener, 1973
).
) and trypomastigotes (Tardieux et al.,
1994
; Burleigh and Andrews, 1995
; Barr et al., 1996
) contain a soluble factor that induces transient increases in the
cytosolic free calcium concentration ([Ca2+]i)1 of a variety
of mammalian cell types (Burleigh and Andrews, 1995
). In
response to live trypomastigotes or trypomastigote soluble extracts, Ca2+ is mobilized from intracellular stores in an
IP3-mediated (Rodriguez et al., 1995
), pertussis toxin-sensitive pathway (Tardieux et al., 1994
). Prevention of these
transients by buffering host cell intracellular free Ca2+
(Tardieux et al., 1994
) or depleting intracellular Ca2+
stores (Rodriguez et al., 1995
) results in inhibition of parasite invasion. Furthermore, rapid rearrangements in the
host cell actin cytoskeleton are observed as a consequence
of trypomastigote-induced Ca2+ transients (Rodriguez et al.,
1995
). Since experimental depolymerization of host cell
actin microfilaments results in enhancement of invasion by
T. cruzi (Schenkman et al., 1991
; Tardieux et al., 1992
), the available evidence supports the postulated role for Ca2+
signaling in facilitating cell invasion by these parasites.
). On the
basis of protease inhibitor profile and substrate specificity,
an 120-kD peptidase was identified in T. cruzi trypomastigotes as a candidate for involvement in mammalian cell
signaling (Burleigh and Andrews, 1995
). However, the purified peptidase had no Ca2+-signaling activity on its own,
and it was found to be present at similar levels in epimastigotes, a noninvasive life cycle stage of T. cruzi, which
does not signal mammalian cells (Burleigh and Andrews,
1995
). Removal of a heparin-binding fraction from the trypomastigote soluble extract resulted in a depletion of Ca2+
signaling but not of peptidase activity, suggesting that, in addition to the peptidase, a factor present only in invasive
forms of the parasite is required for signaling (Burleigh
and Andrews, 1995
). This second component could be a
precursor that is processed by the peptidase, resulting in a
mature Ca2+ agonist. Alternatively, it could function as a
recognition molecule, perhaps involved in targeting the
peptidase to the host cell membrane where its subsequent
activity would result in the induction of Ca2+ transients.
; Kreil,
1990
). Antibodies to the recombinant peptidase inhibit
both peptidase activity and Ca2+ signaling in mammalian
cells by trypomastigote extracts, providing direct evidence
for participation of the T. cruzi oligopeptidase B in this
signaling pathway.
Materials and Methods
). Amastigotes were generated in
culture by incubating freshly harvested trypomastigotes in liver infusion
tryptose medium containing 10% FBS at 37°C, 5% CO2 for 24 h. Epimastigotes (Y strain and Dm28c) were cultured in liver infusion tryptose medium at 28°C (Nogueira and Cohn, 1976
). Metacyclic parasites were purified from stationary phase cultures of epimastigotes (Dm28c) by passing
parasites through a DEAE-cellulose column (Yoshida, 1986
).
), resuspended at 2 × 108 parasites per ml in Dulbecco's PBS containing 2 mM CaCl2 and 1 mM MgCl2
(PBS2+), and 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 2.5. 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 using a Ti50.1 rotor (Beckman Instruments,
Fullerton, CA), and the resulting supernatant fraction was passed over a
Con A-Sepharose column (Pharmacia, Uppsala, Sweden). The unbound
material was then collected, aliquoted, and stored at
80°C.
). Briefly, epimastigotes, grown
and harvested as described above, were resuspended in PBS2+ at a concentration of 5 × 108/ml, heated to 56°C for 5 min, and then immediately
frozen at
80°C. Thawed parasite suspensions were adjusted to 20 µM N-[N- (l-3-trans-carboxirane-2-carbonyl)-l-leucyl]-agmatine (E-64) to inhibit
the T. cruzi major lysosomal protease, cruzain (Cazzulo et al., 1989
), before preparation of the parasite soluble fraction as described above. The
flow-through fraction from Con A-Sepharose (50 ml) was applied to a 10ml column of DEAE-cellulose equilibrated in PBS2+. Unbound material
containing the 120-kD 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 PBS2+ containing 20 µM E-64, filtered through a 0.22-µm filter
unit (Millipore Products Division, Bedford, MA) and desalted on an Econo-Pac® 10DG column (BioRad Laboratories, Hercules, CA) equilibrated in PBS2+. 4 ml of desalted material was applied to a 1.6 cm × 60 cm
Sephacryl-300 column (Pharmacia) equilibrated in PBS2+ and proteins
were eluted in the same buffer, collected as 2-ml fractions. Peptidase activity was assayed using the synthetic substrate Z-phenylalanyl-arginyl-7amino-4-methyl coumarin (Z-Phe-Arg-AMC) (Enzyme Systems Products, Livermore, CA) as described previously (Burleigh and Andrews,
1995
). Peptidase-containing fractions were pooled and concentrated by
ammonium sulfate precipitation (70% saturation). The pellet was resuspended in 25 mM Bis-Tris, pH 6.4, desalted, and applied to a Mono-Q HR
5/5 column (Pharmacia). Bound material was eluted with a gradient of
0-250 mM NaCl, 25 mM Bis-Tris, pH 6.4. The 120-kD enzyme eluted at a
salt concentration of ~75-90 mM NaCl. These fractions were pooled and
applied to a Mono-P HR 5/5 chromatofocusing column (Pharmacia) and
bound material eluted with a pH gradient of 6.4-4.0 formed with Polybuffer 74 (Pharmacia). At this point in the purification scheme, it was possible to detect a significant 120-kD band in Coomassie-stained SDSPAGE gels that hydrolyzed the synthetic substrate Z-Phe-Arg-AMC in a
gel overlay assay (Burleigh and Andrews, 1995
). Thus, the 120-kD band
containing purified peptidase (~10 µg) was excised from an SDS-PAGE
gel and submitted to the W.M. Keck Biotechnology Resource Laboratory
at Yale University for in-gel tryptic digestion and amino acid sequence
analysis of HPLC-purified peptides.
). Degenerate oligonucleotide primers were designed in forward and reverse orientations
based on the amino acid sequence of two tryptic fragments of the peptidase: DYMNSYSPVD and DEPVFWA. These primers were used to amplify DNA from first strand cDNA that was synthesized from 10 µg total
trypomastigote RNA using AMV reverse transcriptase under conditions
suggested by the manufacturer (Boehringer Mannheim Corp., Indianapolis, IN). For PCR amplification of peptidase DNA, 5-µl aliquots of trypomastigote cDNA were used as template, and PCR reactions were carried
out using the GeneAmp kit (Perkin-Elmer Corp., Norwalk, CT) with 35 cycles of: 1-min annealing at 40°C, 1-min extension at 72°C, 1-min denaturation at 94°C, and a final 5-min extension step at 72°C, using a PTC-100
automated thermocycler (MJ Research, Inc., Watertown, MA). One combination of forward and reverse primers generated a 750-bp PCR product,
which was directly cloned into the TA-cloning vector pCRII® (Invitrogen,
San Diego, CA) and sequenced from the T7 and SP6 primer sites in the
plasmid by the W.M. Keck Biotechnology Resource Laboratory to confirm its authenticity. The 750-bp fragment was labeled by incorporation of
[32P]dCTP (Amersham Corp., Arlington Heights, IL) during PCR amplification and used as a probe to screen a T. cruzi amastigote cDNA library constructed in
ZAPII (Stratagene, La Jolla, CA; generously provided by
S. Teixeira and J. Donelson, University of Iowa, Iowa City). Plaques of
positive clones were isolated, and pBluescript plasmids containing cDNAs
were excised from the phage according to manufacturer's specifications
(Stratagene). A clone with the largest insert (2.6-kb EcoRI-XhoI fragment), which contained the full-length peptidase cDNA, was completely
sequenced in both directions by the W.M. Keck Biotechnology Resource
Laboratory at Yale University.
-GCT ACC GTG CTC GAG ATG AAG TGT3
) and reverse (5
-GTG ACA CCC TAA CTC GAG CAA TAA-3
)
primers designed with internal Xho I sites (bold); the initiation codon of
the forward primer is underlined. The PCR product was cloned directly
into pCRII (Invitrogen), and then the 2.2-kb XhoI-XhoI fragment was excised and subcloned into the pET19b expression vector (Novagen, Inc., Madison, WI). Restriction analysis was carried out to ensure correct orientation of the gene, and sequencing from the T7 promoter in the plasmid
demonstrated that the ATG was in frame. The peptidase was expressed as
an NH2-terminal polyhistidine-tagged fusion protein in E. coli BL21 by induction of a log phase culture with 1 mM isopropylthio-
-d-galactoside
for 16 h at 27°C. Bacteria were harvested and the recombinant T. cruzi oligopeptidase (rPEP) was purified on nickel-agarose resin according to the
manufacturer's specifications (Novagen, Inc.).
). For affinity purification of antibodies, purified rPEP
(750 µg) was coupled to 400 µl of ECH-Sepharose resin (Pharmacia) by
addition of 0.1 M of the cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC; Sigma Chemical Co., St. Louis, MO). The
beads were washed and unreacted groups were blocked with 100 mM ethanolamine for 1 h at 37°C. The rPEP-Sepharose was washed extensively
with dH2O and equilibrated in 10 mM Tris-HCl, pH 7.5, before incubating overnight at 4°C with antipeptidase IgG (5 ml of IgG at 1 µg/ml). Unbound IgG was removed by washing the rPEP-Sepharose column with 10 ml of 10 mM Tris-Cl, pH 7.5, followed by 10 ml of 500 mM NaCl, 10 mM Tris-Cl, pH 7.5. Antipeptidase IgG was eluted with 5 ml 100 mM glycine, pH 2.5, and neutralized with 500 µl 1 M Tris-HCl, pH 8.0.
) containing 5%
-2-mercaptoethanol at 25° or
100°C for 5 min before separation on 7.5% polyacrylamide gels. Proteins
were transferred to ImmobilonTM membranes (Millipore Corp.) according
to the method of Towbin et al. (1979)
. After an overnight incubation at
4°C in blocking buffer (5% nonfat milk, 20 mM Tris, pH 7.4, 15 mM NaCl,
0.05% sodium azide), blots were incubated with 25 µg/ml guinea pig anti- peptidase IgG or 5 µg/ml rabbit anti-rPEP IgG in blocking buffer for 2 h
at room temperature. Blots were washed five times with 20 mM Tris, pH
7.4, 150 mM NaCl, and 0.1% Tween 20 (TBST) for 5 min each wash, and
then incubated with a 1:4,000 dilution of alkaline phosphatase-labeled
goat anti-guinea pig IgG or goat anti-rabbit IgG (Kirkegaard & Perry
Laboratories, Gaithersburg, MD) for 1 h in blocking buffer. After a 30min wash with TBST, immunoreactive bands were detected by incubating
blots with the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl1-phosphate/nitro blue tetrazolium (BCIP/NBT; Promega Corp., Madison, WI).
). For kinetic analysis, rPEP was
diluted with 50 mM Tris-Cl, pH 8.0, 150 mM NaCl to a final concentration
of 6 × 10
9 M, and substrates were added in the range of 2-15 µM. Initial velocity (vo) of substrate hydrolysis was recorded as the release of AMC
over 60 s (during which the rate of AMC hydrolysis was linear) in a model
F-2000 spectrofluorimeter (Hitachi Instruments Inc., Danbury, CT) at excitation and emission wavelengths of 380 and 440 nm, respectively. Km values were calculated from Lineweaver-Burke plots (Segel, 1976
). Inhibitor
sensitivity was carried out by incubation of rPEP (5 ng) in 500 µl with
varying concentrations of inhibitors at room temperature for 10 min before adding to 1.5 ml 10 µM Z-Phe-Arg-AMC in TBS and measuring hydrolysis of substrate for 5 min in the spectrofluorimeter, as described
above.
-ATGAAGTGTGGTCCCATTGCCACG-3
; reverse primer: 5
-CATTTCCACCTCCGAAGAAGTGTC-3
) designed to produce a full-length amplification product.
Amplification conditions were as follows: 40 cycles of 1 min at 94°C; 1 min at
50°C; 3 min at 72°C; and a final step of 7 min at 72°C. Southern blot analysis of PCR products separated by electrophoresis on a 1% agarose gel was
carried out to determine the specificity of the resulting 2.1-kb bands. Blots
were hybridized with the 2.5-kb EcoRI-XhoI fragment excised from the
full-length cDNA clone. The EcoRI-XhoI fragment was purified from a
0.7% agarose gel using the CompassTM DNA purification kit (American Bioanalytical, Natick, MA), and 50 ng was used in a random priming reaction
(Boehringer Mannheim Corp.) with 50 µCi[32P]dCTP (Amersham Corp.).
Hybridization was carried out overnight at 42°C in 50% formamide, 6×
SSC, 0.5% SDS, and 5× Denhardt's solution. The blots were washed to a
final stringency of 0.6× SSC at 55°C before exposure to X-OMat film
(Eastman Kodak Co., Rochester, NY).
). To determine the
number of genomic copies of the T. cruzi peptidase gene, 5 µg aliquots of
total genomic DNA were digested for 16 h with restriction enzymes, and
digested DNA was separated on 0.9% agarose gels in 0.5× Tris-borateEDTA buffer and transferred to Hybond-N membranes by capillary blotting in 20× SSC for 16 h. Blots were hybridized with the full-length peptidase cDNA probe.
). Ca2+-signaling activity of trypomastigote soluble extracts was also assayed at the single cell level, by time lapse fluorescence video microscopy. NRK cells were
plated 48 h before the assay at 1.3 × 105 per dish, on 3.5-cm glass bottom
microwells (MatTek Corp., Ashland, MA), and loaded with 5 µM fluo3/AM (Molecular Probes Inc.) for 45 min at 37°C. After loading, the
dishes were washed in Hepes-buffered Ringer's solution (Heuser, 1989
)
and placed on the heated stage of an Axiovert 135 microscope (Carl Zeiss,
Inc., Thornwood, NY). Fluorescence images were collected with a video
camera (CCD-C72; Dage/MTI, Michigan City, IN) through a ×40 objective and recorded on an optical memory disk (TQ-3031F; Panasonic, Secaucus, NJ), at time-lapse intervals of 1 s using a computer-controlled
shutter system (Metamorph software; Universal Imaging Corp.,
Westchester, PA). Parasite soluble extracts (50 µl) preincubated with preimmune or immune anti-rPEP IgG (200 µg/ml) were added to the cells at 50 s after initiation of the time-lapse recording. Pseudocolored images
were transferred to Adobe Photoshop (Adobe Systems, Inc., Mountain
View, CA), composed and printed in a high resolution color printer (XLS
8600PS; Eastman Kodak Co.).
) using affinity-purified antipeptidase IgG at 20 µg/ml and 10 nm protein A-gold. Micrographs were taken using a 410 TEM (Phillips Instrument Co., Marwar, NJ) operating at 80 kV.
Results
).
The 120-kD band, shown to have peptidase activity in a gel
overlay assay (Burleigh and Andrews, 1995
), was excised
from an SDS-PAGE gel and subjected to trypsin digestion, peptide purification by HPLC, and amino acid sequencing of selected peptides. Degenerate oligonucleotides designed based on amino acid sequences of internal
tryptic peptides of the 120-kD peptidase were used in reverse transcriptase (RT)-PCR reactions with total cellular RNA from trypomastigotes as template. The resulting
750-bp PCR product was cloned, sequenced from both ends, and found to contain homologous sequences to
known peptidases by searching the available sequence databases. This 750-bp PCR product was used as a probe to
screen a T. cruzi cDNA library, from which several positive clones were obtained. The clone with the largest insert
(2.6 kb) was completely sequenced in both directions (GenBank accession number U69897) and a 2215-bp open
reading frame was identified that contained an A-T rich
region with a stop codon (TAA) 42 bp upstream of the
ATG designated as the initiation codon (Fig. 1). Several
other clones containing smaller inserts (2.1 and 1.4 kb)
were partially sequenced and found to be peptidase cDNA
clones lacking complete 5
ends (not shown).
Fig. 1.
Nucleotide sequence of cloned cDNA and
deduced amino acid sequence of the T. cruzi oligopeptidase B. The deduced
amino acid sequence is
shown below the nucleotide
sequence. Both are numbered on the right. Amino
acid sequences obtained for
tryptic peptides of the purified peptidase (underlined).
The stop codons that limit the open reading frame
(bold); polyadenylation signals are highlighted (double
underline). These sequence
data are available from GenBank under accession number U69897.
[View Larger Version of this Image (70K GIF file)]
). However, because the T. cruzi peptidase was shown to migrate in an SDS-PAGE gel as a band of 120 kD, it was
of interest to determine the relationship between the 81-kD
polypeptide predicted by the cDNA sequence and the native molecule. To accomplish this, polyclonal antibodies
raised to gel-extracted T. cruzi 120-kD peptidase were
used to probe Western blots of trypomastigote soluble proteins and rPEP separated by SDS-PAGE under nonreducing and reducing conditions. Immunoreactive bands of
~120 and 80 kD were detected in both the soluble trypomastigote extract and purified rPEP under nonreducing
conditions (Fig. 2 A, lanes 1). However, when samples
were fully reduced and boiled, the 120-kD band was no
longer detectable and the 80-kD one was the major band
(Fig. 2 A, lanes 2). The two lower molecular weight bands
observed in reduced boiled samples are likely to be degradation products, since they occur in both the native enzyme and rPEP samples (Fig. 2 A, lanes 2). These results
demonstrate that the T. cruzi oligopeptidase B, both parasite derived and recombinant, migrates with an apparently higher molecular weight on SDS-PAGE gels when electrophoresis is carried out under conditions known to preserve peptidase activity (Burleigh and Andrews, 1995
), but
migrates at the expected size of 80 kD when fully denatured.
Fig. 2.
Molecular size determination of native and recombinant T. cruzi oligopeptidase B. (A) Immunoblots of oligopeptidase B partially purified from epimastigotes (T. cruzi, lanes 1 and 2)
or 0.6 µg purified recombinant peptidase (rPEP, lanes 1 and 2)
with guinea pig polyclonal anti-oligopeptidase B IgG (25 µg/ml)
followed by acid phosphatase-conjugated anti-guinea pig IgG.
Samples were reduced with 5% -mercaptoethanol and kept at
either 25°C (lanes 1) or heated for 5 min at 100°C (lanes 2) before
SDS-PAGE. (B) FPLC fractionation of a trypomastigote soluble
extract on Sephacryl-300. Proteins were eluted in PBS2+ while
A280 nm was continuously monitored (open squares). The peak
absorbance value (fraction 41) was taken as 100% and the baseline as 0% protein. Peptidase activity (black circles) was assayed
in 20 µl aliquots of each fraction using ZFR-AMC as a substrate,
as described in Materials and Methods. Fractions containing peptidase activity (64-76) were pooled, concentrated, reduced, boiled
for 5 min, separated by SDS-PAGE, and immunoblotted with anti-oligopeptidase B guinea pig IgG as described above. The
elution of molecular mass standards is indicated by letters (a) catalase = 232 kD; (b) aldolase = 158 kD; (c) albumin = 67 kD; (d)
ribonuclease A = 13.7 kD.
[View Larger Version of this Image (23K GIF file)]
; Santana et al., 1992
; Burleigh and Andrews, 1995
).
Kinetic analyses using purified rPEP were carried out with
three Arg-containing synthetic substrates. The calculated
Km values for these substrates were similar: Z-Phe-ArgAMC and Z-Gly-Gly-Arg-AMC had Km values of 6 ± 2 µM,
and Z-Arg-Arg-AMC of 5 ± 1 µM. No hydrolysis of Z-GlyPro-AMC, Z-Gly-Phe-AFC, or Z-Ala-AFC by the recombinant peptidase was detected, indicating specificity for
cleavage at the carboxyl side of arginine residues, as previously reported (Ashall, 1990
; Santana et al., 1992
; Burleigh
and Andrews, 1995
). Sensitivity of the recombinant oligopeptidase B to several different protease inhibitors was then examined. The concentration of inhibitor that resulted in a 50% reduction of the initial rate of hydrolysis of
Z-Phe-Arg-AMC (IC50) was determined: 0.02 µM leupeptin, 0.05 µM antipain, 0.33 µM p-chloromercuribenzoic
acid, 0.6 µM N-
-p-tosyl-l-lysine-chloromethyl ketone,
10 µM chymostatin, 225 µM diisopropyl fluorophosphate, 0.25 µM Z-Phe-Arg-fluoromethylketone, and 0.2 µM acetylArg-Arg-chloromethylketone. Millimolar concentrations
of PMSF, aprotinin,
2-macroglobulin, EDTA, and E-64
did not inhibit recombinant oligopeptidase B activity.
), thereby confirming the authenticity of the cDNA clone and indicating that it contains the entire open reading frame (Fig. 1).
) that harbors the active site serine residue in all members of this enzyme family was also found in the
T. cruzi sequence (Fig. 3). Since the greatest degree of homology between members of the prolyl oligopeptidase
family has been shown to occur within the COOH-terminal third of the molecule (Barrett and Rawlings, 1992
),
this region of the T. cruzi peptidase was aligned with the
protease II from E. coli (Kanatani et al., 1991
) and Moraxella lacunata (Yoshimoto et al., 1995
), and prolyl endopeptidases from Flavobacterium meningosepticum (Yoshimoto et al., 1991
), Aeromonas hydrophila (Kanatani et al.,
1993
), and porcine brain (Rennex et al., 1991
). From the
partial sequence alignment (Fig. 3), it is apparent that
these molecules share significant homology, particularly in
the vicinity of the active site serine residues (Fig. 3, asterisk). The T. cruzi peptidase was more similar overall to the
Moraxella and E. coli peptidases, having 34% and 30%
identical residues, respectively. Less identity to the T. cruzi
enzyme was observed with peptidases from Flavobacterium (22%), Aeromonas (21%), and pig brain (18%). The
T. cruzi sequence contains conserved Asp and His residues at positions 647 and 682, which have been proposed
to form the catalytic triad with Ser in this family of enzymes (Stone et al., 1991
; Yoshimoto et al., 1995
). It was
on the basis of sequence similarity with the Moraxella and
E. coli protease II enzymes (since renamed oligopeptidase B;
Tsuru and Yoshimoto, 1994
) that the T. cruzi enzyme was
named oligopeptidase B.
Fig. 3.
The T. cruzi oligopeptidase B is homologous
to members of the prolyl oligopeptidase family of serine
hydrolases. Residues 412-
683 of the T. cruzi oligopeptidase B (top) are aligned with Moraxella lacunata (residues
385-655; Yoshimoto et al.,
1995) and Escherichia coli
protease II (residues 384-
653; Kanatani et al., 1991
),
and prolyl oligopeptidases
from Flavobacterium meningosepticum (residues 409-
676; Yoshimoto et al., 1991
),
Aeromonas hydrophila (residues 390-657; Kanatani et
al., 1993
), and porcine brain (residues 403-681; Rennex et al., 1991
). Identical residues are boxed, and the active site serine residue included in the consensus sequence GxSxGGzz is denoted by an asterisk (*).
[View Larger Version of this Image (58K GIF file)]
Fig. 4.
The T. cruzi oligopeptidase B gene is present
as a single copy in the genome and expressed in all
life cycle stages of the parasite. (A) Southern blot of T. cruzi genomic DNA digests
probed with the full-length
oligopeptidase B cDNA labeled with [32P]dCTP. The
blot was washed to a final
stringency of 0.6× SSC at
60°C and exposed to film for
5 d. (B) Expression of the
oligopeptidase B gene in life
cycle stages of T. cruzi. (I)
Amplification of homologous
sequences from total parasite
RNA by RT-PCR. PCR
products were separated on
agarose, blotted to nylon,
and probed with the fulllength peptidase cDNA, revealing a single band of 2.1 kb in three life cycle stages of
T. cruzi. (II) Detection of
peptidase activity in parasite
soluble extracts separated by
SDS-PAGE. Total soluble
extract was prepared from
each parasite life cycle stage, and 3 × 107 parasite equivalents were loaded in each lane. Oligopeptidase B activity was detected directly in a gel overlay assay as described in the Materials and
Methods. Active purified recombinant peptidase (0.6 µg; rPEP) migrated with an apparent molecular mass of 120 kD, similar to oligopeptidase B activity expressed in each developmental stage of T. cruzi: epimastigotes (Epi), metacyclics (Meta), amastigotes (Amast),
and trypomastigotes (Trypo).
[View Larger Version of this Image (35K GIF file)]
). To determine whether the peptidase gene was expressed in other life cycle stages of T. cruzi,
RT-PCR was carried out using total RNA prepared from
each stage, and specific oligonucleotide primers predicted
to result in the amplification of a full-length PCR product.
PCR products were separated on agarose gels, blotted, and probed with a full-length cDNA probe (Fig. 4, B-I)
that revealed a single 2.1-kb band present in the three
main life cycle stages of T. cruzi. For direct detection of
oligopeptidase B activity in the life cycle stages of the parasite, soluble extracts were prepared and separated on
SDS-PAGE gels, and peptidase activity was detected in a
gel overlay assay using Z-Phe-Arg-AMC as the substrate.
As shown in Fig. 4, B-II, peptidase activity migrating at an
apparent molecular mass of 120 kD was found in all parasite life cycle stages. The 120-kD band was previously
shown to be the only peptidase detectable with this substrate in Con A-unbound soluble fractions of T. cruzi (Burleigh and Andrews, 1995
). Purified recombinant oligopeptidase B was also included in the gel (Fig. 4, B-II, rPEP) to
demonstrate that the active product of the cloned peptidase gene expressed in E. coli also migrates at 120 kD,
identical to the enzyme isolated from T. cruzi (Fig. 2 A;
Burleigh and Andrews, 1995
).
rPEP). Additional bands of lower intensity appearing on this blot are also recognized by the
same concentration of IgG prepared from preimmune rabbit sera (Fig. 5 B, Pre), with the exception of a band of
~40 kD that is most likely an oligopeptidase B degradation product.
Fig. 5.
Antibodies to the recombinant peptidase inhibit peptidase activity and Ca2+ signaling in mammalian cells by trypomastigote extracts. (A) Oligopeptidase B activity in TSE pretreated with
increasing concentrations of preimmune rabbit IgG (open circles)
or immune rabbit IgG against rPEP (black circles). Purified IgG
ranging from 0.75-500 µg/ml was added to TSE followed by a 1-h
incubation at 4°C. Hydrolysis of the Z-Phe-Arg-AMC oligopeptidase B substrate was measured at 37°C for 10 min, as described in
Materials and Methods. The values for nmol AMC released are
plotted as a function of IgG concentration ([IgG] µg/ml). (B) Total trypomastigote soluble extract (6 × 106 parasite equivalents)
was heated to 100°C in Laemmli sample buffer in the presence of
5% 2-mercaptoethanol before separation on a 10% SDS-PAGE
gel and immunoblotting with 5 µg/ml
rPEP (
rPEP) or 5 µg/ml
IgG prepared from the preimmune rabbit serum (Pre) as described in the Materials and Methods. The arrow indicates the
80-kD oligopeptidase B. (C) Effect of anti-rPEP on the Ca2+-signaling activity of TSE on NRK fibroblasts. TSE was preincubated with 200 µg/ml preimmune IgG (black line) or anti-rPEP IgG (grey line) for 1 h at 4°C. The ability of TSE to induce [Ca2+]i transients
in NRK cells loaded with the Ca2+-sensitive dye fura-2/AM was
measured in a spectrofluorometric assay, as described in Materials and Methods. Results are plotted as the ratio of the fluorescence emission of fura-2/AM at 510 nm after alternating excitation at 340 nm/380 nm against time (s). TSE was added to the
cells at the points indicated by the arrow. The results of six independent experiments are shown.
[View Larger Version of this Image (24K GIF file)]
; Burleigh and
Andrews, 1995
), TSE was pretreated with either preimmune or immune IgG at a final concentration of 200 µg/ml, and then added to fura-2/AM-loaded NRK fibroblasts.
Preincubation of TSE with anti-rPEP antibodies resulted
in a significant delay in the induction of cytosolic free Ca2+
transients, when compared with treatment with preimmune IgG (Fig. 5 C). In addition, the level of the response
was significantly reduced in four out of six experiments
(Fig. 5 C).
; Burleigh
and Andrews, 1995
), the recombinant peptidase had no
Ca2+-signaling activity by itself when added to NRK cells
at a specific activity equivalent to trypomastigote extracts
(not shown).
Fig. 6.
Antibodies to the recombinant T. cruzi oligopeptidase B delay the onset of [Ca2+]i transients in NRK cells. Time-lapse fluorescence microscopy images of NRK cells loaded with the Ca2+-sensitive dye fluo-3/AM. Images shown were acquired 40 s before, and
20, 30, 50, and 100 s after exposure to TSE. In the two experiments shown on the top of the figure, TSE was preincubated for 1 h at 4°C
with 200 µg/ml preimmune rabbit IgG (PI); in the two experiments shown at the bottom, preincubation was carried out with 200 µg/ml anti-rPEP immune IgG (I). Fluorescence intensity is pseudocolored, with the highest [Ca2+]i shown in red and the lowest in blue. Bar, 10 µm.
[View Larger Version of this Image (147K GIF file)]
T. cruzi Oligopeptidase B Is Localized in the Cytoplasm of Trypomastigotes
The cDNA sequence of the T. cruzi oligopeptidase B predicts a soluble, hydrophilic protein with no secretion signal
sequence (Fig. 1). Consistent with this finding, >90% of
oligopeptidase B is released in soluble form and found in
the supernatant of 100,000 g centrifugations when trypomastigotes are disrupted by sonication (not shown). For
determining the precise localization of the enzyme in the
parasites, ultrathin cryosections of aldehyde-fixed trypomastigotes and amastigotes were labeled with affinity-
purified guinea pig anti-oligopeptidase B IgG, followed by
10 nm protein A-gold. Labeling was observed throughout
the cells, and appeared to be restricted to the cytosol in
both trypomastigotes (Fig. 7 A) and amastigotes (Fig. 7 B).
No labeling was detected on the surface of the parasites or
in the flagellar pocket. In addition, no label was associated
with organelles such as the ER, Golgi complex, nucleus, or
kinetoplast. This pattern of labeling is consistent with a cytoplasmic localization.
The unusual invasion mechanism recently elucidated for
T. cruzi, which involves recruitment and fusion of host cell
lysosomes at the parasite entry site (Tardieux et al., 1992;
Rodriguez et al., 1996
), led us to search for signal transduction events triggered specifically by the forms capable
of invading mammalian cells. A soluble factor that generates IP3 and releases Ca2+ from the intracellular stores of
mammalian cells was detected and found to be present
only in the infective trypomastigote and metacyclic life cycle stages (Tardieux et al., 1994
; Burleigh and Andrews,
1995
; Dorta et al., 1995
; Barr et al., 1996
). Buffering of
host cell free intracellular Ca2+ (Tardieux et al., 1994
) or
depletion of intracellular Ca2+ stores (Rodriguez et al.,
1995
) blocked trypomastigote entry, suggesting that the
Ca2+-signaling activity, detected also with intact parasites,
is essential for invasion.
Results from a previous study using protease inhibitors
(Burleigh and Andrews, 1995) suggested that the activity of
a T. cruzi 120-kD peptidase was coupled to the Ca2+-signaling activity of trypomastigotes. Here, we describe the cloning of the gene encoding this peptidase, whose sequence revealed its relationship to a family of serine oligopeptidases that includes hormone-processing enzymes
(Fuller et al., 1988
; Kreil, 1990
). Most importantly, using
specific antibodies generated against the recombinant
T. cruzi oligopeptidase B, we provide direct evidence for
involvement of this enzyme in the generation of Ca2+ transients in mammalian cells by trypomastigote extracts. Our present results are consistent with the hypothesis that the
T. cruzi oligopeptidase B, although not capable of signaling by itself, is required for generating a Ca2+-signaling
factor for mammalian cells (Burleigh and Andrews, 1995
).
The T. cruzi oligopeptidase B shares significant sequence homology with members of the recently defined
prolyl oligopeptidase family (Barrett and Rawlings, 1992).
This family of serine hydrolases contains both endo- and
exopeptidases, soluble and membrane-associated enzymes
that exhibit variable but quite restricted substrate specificities. Prolyl oligopeptidases contain a consensus sequence
GxSxGGzz that includes the active site serine residue, and
they have a proposed mechanism of catalysis involving a
Ser-Asp-His charge relay system (Barrett and Rawlings,
1992
). The highest degree of sequence conservation among
these enzymes, including the T. cruzi oligopeptidase B, is
observed within the COOH-terminal third of the molecule, where the catalytic residues are situated. The T. cruzi
oligopeptidase B is most similar to the Moraxella and E. coli
peptidases in terms of primary sequence and substrate
specificity, cleaving substrates on the COOH-terminal side
of basic amino acids (Pacaud and Richaud, 1975
; Yoshimoto et al., 1995
). Less identity was found between the T. cruzi enzyme and the Flavobacterium, Aeromonas, and pig brain peptidases, all of which are post-prolyl cleaving enzymes (Rennex et al., 1991
; Yoshimoto et al., 1991
; Kanatani et al., 1993
). As additional peptidases containing the
GxSxGGzz consensus sequence are identified, a division
in the prolyl oligopeptidase family may emerge, based on
sequence identity as well as substrate specificity. Since the
T. cruzi oligopeptidase B is the first protozoan member of
the prolyl oligopeptidase family to be cloned and sequenced, it would be interesting to determine if similar peptidases are present in other ancient eukaryotes. The
extensive sequence similarity observed between the T. cruzi
oligopeptidase B and the bacterial enzymes was somewhat
surprising, although an additional example of a closer relationship of a trypanosome gene with bacterial but not eukaryotic homologues has recently appeared in the literature (Stebeck et al., 1996
).
Physiological substrates have been identified for only a
few members of the prolyl oligopeptidase family of serine
peptidases. Some of them, such as the human dipeptidyl
aminopeptidase IV and aminopeptidase P, were shown to
cleave biologically active peptides in vitro, which led to a
proposed role for these enzymes in the final degradation
of mature peptide agonists (Mentlein, 1988). However,
there is significant evidence that members of this family also participate in the final steps of maturation of regulatory peptides (Fuller et al., 1988
; Mentlein, 1988
; Kreil, 1990
).
One of the best characterized examples is the Saccharomyces cerevisiae dipeptidyl aminopeptidase A (DPAP A),
which is involved in the maturation of the yeast mating
hormone
-factor (Fuller et al., 1988
). DPAP A is a transGolgi enzyme that removes dipeptides from the NH2 terminus of pro-
-factor to release the 17-amino acid active
hormone as the last step in the
-factor processing cascade (Fuller et al., 1988
). Studies of substrate preferences for
prolyl oligopeptidases have shown that these enzymes
preferentially hydrolyze small peptides with an upper limit
of 30 amino acids, and therefore have been described as
"true oligopeptidases" (Barrett and Rawlings, 1992
; Polgar, 1992
). A notable exception is the E. coli oligopeptidase B, which was shown to hydrolyze aspartokinase I-homoserine dehydrogenase at a hinge region separating the two
functional domains of the polypeptide (Veron et al., 1972
;
Pacaud and Richaud, 1975
). However, cleavage of this
protein by the E. coli peptidase does not occur in the presence of threonine, an allosteric effector for the dehydrogenase, suggesting that in larger substrates conformational
changes may be required for the exposure of bonds susceptible to cleavage.
Purification and characterization of a high molecular
weight alkaline peptidase from T. cruzi that hydrolyzes
substrates on the carboxyl side of basic amino acid residues was reported previously, although no biological role
was proposed for it (Ashall, 1990; Santana et al., 1992
).
Based on the reported characteristics, it is likely that the
enzyme described in those studies is the same as the one
we have now cloned and expressed. Expression of the fulllength T. cruzi oligopeptidase B cDNA in E. coli resulted in the overproduction of active peptidase with identical
properties to the enzyme purified from soluble parasite
extracts (Burleigh and Andrews, 1995
). Both enzymes migrated in SDS-PAGE gels at an apparent molecular mass
of 120 kD when active, but were reduced to the predicted
size of 80 kD when fully denatured. On gel filtration columns, the enzymes eluted with a molecular mass fraction
of ~180 kD, suggesting dimerization.
Polyclonal antibodies directed to the purified active recombinant peptidase were effective in blocking the peptidase activity in trypomastigote soluble extracts and also
resulted in inhibition of the Ca2+ transients elicited in
NRK fibroblasts by this fraction. Under conditions where
90% of the oligopeptidase B activity was inhibited, the
generation of Ca2+ transients was significantly delayed
and often reduced in intensity, an observation that was
made while measuring the transient increase of cytosolic
free Ca2+ in a population spectrofluorimetric assay, and
also at the single cell level. Our previous results, which
strongly implicated a role for oligopeptidase B in the signaling mechanism (Burleigh and Andrews, 1995), have
now been confirmed. The results presented here provide
direct evidence for the involvement of the T. cruzi oligopeptidase B in generating Ca2+ transients in mammalian cells.
Purified oligopeptidase B was unable to elicit Ca2+ transients in mammalian cells (Burleigh and Andrews, 1995),
and, similarly, the recombinant oligopeptidase B had no
effect. It was previously demonstrated that removal of a
heparin-binding fraction from the trypomastigote soluble
extract resulted in depletion of Ca2+ signaling activity,
whereas oligopeptidase B activity persisted (Burleigh and
Andrews, 1995
). It was therefore postulated that a factor(s) expressed only in invasive forms of T. cruzi would be processed by oligopeptidase B, as an obligatory step to
generate an active Ca2+ agonist for mammalian cells. Assuming that the antibodies against the recombinant oligopeptidase B directly interfere with activity of the native
peptidase toward its natural substrate, as indicated by the
strong inhibition observed on hydrolysis of synthetic peptide substrates, the delayed Ca2+ response observed in the
presence of anti-rPEP IgG may reflect a slower rate of agonist generation by the residual peptidase activity (10%).
We reported previously that serial dilutions of trypomastigote soluble extracts result in proportionately decreased Ca2+-signaling responses in mammalian cells (Burleigh and
Andrews, 1995
). Here we show that after a 90% reduction
of oligopeptidase B activity in parasite extracts, Ca2+ transients still occur, albeit at a slower rate and reaching lower
levels. This is consistent with a scenario in which the substrate is the limiting factor, with the peptidase being required only in small amounts as a result of continuous
turnover during the catalytic process.
The pattern of Ca2+ signaling induced in mammalian
cells by intact trypomastigotes is asynchronous and repetitive (Tardieux et al., 1994), in contrast with parasite-soluble extracts that trigger a major simultaneous Ca2+ transient in all cells of the population (Burleigh and Andrews, 1995
). This suggests that the processed trypomastigote signaling factor may be released from live parasites in small
amounts and delivered to host cells in a localized fashion.
This scenario is consistent with the recent demonstration
that recruitment of host cell lysosomes, a process proposed
to be regulated by [Ca2+]i transients, is highly localized, involving only lysosomes located at ~10 µm from the parasite invasion site (Rodriguez et al., 1996
).
The postulated role of the T. cruzi oligopeptidase B in
the processing of an immature agonist of trypomastigotes
is consistent with its structural relationship to enzymes
with a very restricted substrate specificity, including the
yeast DPAP A that has a known role in prohormone processing. The lack of a secretion signal sequence and the cytosolic localization of the T. cruzi oligopeptidase B imply
that the postulated processing steps must occur in the cytoplasm of the parasites, with the putative active factor being released by an unconventional mechanism. An alternative pathway for the extracellular release of polypeptides
lacking classical secretory signal sequences is emerging
(Cooper and Barondes, 1990; Muesch et al., 1990
; Rubartelli et al., 1992
; Florkiewicz et al., 1995
; Siders and Mizel,
1995
). Some of the proteins in this group have clearly defined signaling roles outside cells, for example, interleukin
(IL)-1
(Siders and Mizel, 1995
) and basic FGF (Florkiewicz et al., 1995
). In the case of IL-1
, conversion from
pro-IL-1
to the active IL-1
is known to be mediated by
a cytosolic cysteine protease, ICE (Kostura et al., 1989
;
Cerretti et al., 1992
). Little is known about the export
mechanism of cytoplasmically processed polypeptides, except that agents that perturb Golgi function do not block
it, and that vesiculation of the plasma membrane has been
implicated in some cases (Cooper and Barondes, 1990
;
Florkiewicz et al., 1995
). A recent report identified genes
involved in nonclassical protein export in yeast (Cleves et
al., 1996
), and at least one of these genes resembles small
bacterial membrane proteins that function as transporters
(Lewis, 1994
). Now that we have determined that the T. cruzi oligopeptidase B, involved in triggering Ca2+ signaling in mammalian cells, is cytosolic, a search for the processed factor and its release mechanism may provide useful new information about this intriguing process.
Received for publication 17 September 1996 and in revised form 13 November 1996.
This work was funded by National Institutes of Health grant R01AI32056 to N.W. Andrews. B. Burleigh was supported by a postdoctoral fellowship from the Medical Research Council of Canada.We acknowledge the contribution to this work by members of the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University School of Medicine, for carrying out protein microsequencing, oligonucleotide synthesis, and DNA sequencing. We are grateful to S. Teixeira and J. Donelson for providing the T. cruzi cDNA library, A.K. Ma for excellent technical assistance, H. Tan for photography, L. Iadorola and L. Hartnell for EM, A.M. Quinn for assistance with sequence alignments, and C. Tschudi and E. Ullu for helpful discussions.
AFC, 7-amino-4-trifluoromethyl coumarin; AMC, 7-amino-4-methyl coumarin; Z-Phe-Arg-AMC, Z-phenylalanyl-arginyl-7-amino-4-methyl coumarin; [Ca2+]i, intracellular free calcium concentration; E-64, N-[N-(l-3-trans-carboxirane-2-carbonyl)-l-leucyl]- agmatine; IL, interleukin; NRK, normal rat kidney; rPEP, recombinant T. cruzi oligopeptidase B; RT, reverse transcriptase; TSE, trypomastigote soluble extract.