From the Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040
Received for publication, August 11, 2000
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ABSTRACT |
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Cathepsin B-like cysteine protease genes
(cbls) constitute large multigene families in parasitic and
nonparasitic nematodes. Although expressed in the intestine of some
nematodes, the biological and biochemical functions of the CBL proteins
remain unresolved. Di- and tetra-oligopeptides were used as fluorogenic
substrates and irreversible/competitive inhibitors to establish CBL
functions in the intestine of the parasitic nematode Haemonchus
contortus. Cysteine protease activity was detected against
diverse substrates including the cathepsin B/L substrate FR, the
caspase 1 substrate YVAD, the cathepsin B substrate RR, but not the
CED-3 (caspase 3) substrate DEVD. The pH at which maximum activity was
detected varied according to substrate and ranged from pH 5.0 to 7.0. Individual CBLs were affinity isolated using FA and YVAD substrates. pH
influenced CBL affinity isolation in a substrate-specific manner that
paralleled pH effects on individual substrates. N-terminal sequencing
identified two isolated CBLs as H. contortus GCP-7 (33 kDa)
and AC-4 (37 kDa). N termini of each began at a position consistent
with proregion cleavage and protease activation. Isolation of the GCP-7
band by each peptide was preferentially inhibited when competed with a
diazomethane-conjugated inhibitor, Z-FA-CHN2, demonstrating one functional difference among CBLs and among inhibitors.
Substrate-based histological analysis placed CBLs on the intestinal
microvilli. Data indicate that CBLs are responsible for cysteine
protease activity described from H. contortus intestine.
Results also support a role of CBLs in nutrient digestion.
Cathepsin B-like cysteine protease
(cbl)1 genes occur
as large multigene families in a wide range of parasitic and
free-living nematodes (1-3). The functions of CBL proteins remain
unresolved at both a biochemical and biological level. Several
cbl genes were shown to be expressed in the intestine of the
parasitic worm Haemonchus contortus or in the free-living
nematode Caenorhabditis elegans (1, 4). Accordingly, one
function of nematode CBLs might be nutrient digestion.
CBLs may have application to control of parasitic nematodes in multiple
ways. For instance, CBLs that function in host blood digestion are
considered potential anthelmintic targets in schistosome flatworms (5,
6). CBLs are also currently considered as vaccine candidates for immune
control of parasitic nematodes, including H. contortus
(6-9). Alternatively, digestive proteases may indirectly mediate
anthelmintic efficacy involving benzimidazole anthelmintics (10).
Following fenbendazole treatment of lambs, anterior intestinal cells of
H. contortus undergo fragmentation of nuclear DNA and tissue
disintegration (10). The pattern of DNA fragmentation resembled that
associated with apoptosis, which can be initiated by caspase and
noncaspase cysteine proteases (11, 12), while CED-3 is a prominent
nematode caspase (13). Both tissue disintegration and DNA fragmentation
was associated with inhibited transport of secretory vesicles and
subsequent cytoplasmic dispersal of vesicle contents in anterior
intestinal cells (10). The monoclonal antibody (mAb 42/10.6.1) used to monitor secretory vesicle contents binds to a periodate-sensitive determinant found on numerous intestinal membrane and secretory proteins (14). Proteins that were immunoaffinity isolated by this mAb
included an intestinal CBL, among other proteases (15). Therefore, CBLs
might mediate some of the intestinal pathology induced by fenbendazole treatment.
Despite these connections with basic aspects of parasite biology, CBLs
remain enigmatic. The complexity of the family (16) has hindered
biochemical and functional characterization of CBLs. Intestinal CBLs
have predicted signal peptides and are expected to be secreted. CBLs
also have predicted proregions, and N termini of some isolated CBLs
indicated propeptide cleavage (4, 16, 17). Cysteine protease activity
was detected in H. contortus intestine and
excretory-secretory products (ESP). This activity was characterized as
cathepsin L-like, based on hydrolysis of Phe-Arg, but not
Arg-Arg, dipeptide substrates (18). Glu245 was implicated
in determining the ability of cathepsin B to degrade substrates with an
Arg in the P2 position, a characteristic that is distinct from
cathepsins L and S (16, 19). Most predicted CBL sequences from H. contortus lack a Glu corresponding to position 245 in cathepsin B
(16), which theoretically could compromise the ability of H. contortus CBLs to hydrolyze Arg-Arg substrates.
This simple picture is complicated by several observations. In H. contortus ESP, distinct zymogram bands of cysteine protease activity were restricted to acidic pH, while others were active from
acidic to neutral conditions (20). Phe-Arg substrate-based affinity
probes identified four protease bands from ESP. This number was more
restricted than expected according to the size of the CBL gene family
reported. Substantial amino acid diversity was observed among CBL
sequences at multiple positions within predicted S2 and S2' subsite
residues, and then, between CBLs and cathepsin B (16, 19). Such
diversity might translate into CBL properties that are distinct from
related mammalian enzymes. Specific properties have not been attributed
to any individual H. contortus CBL, which has hindered more
directed research on recombinant forms of these enzymes.
In this study, H. contortus intestinal CBLs were directly
linked to known cysteine protease activity from this worm. Substrates for known cathepsin L, cathepsin B, caspase 1, and CED-3 (caspase 3)
enzymes proved effective for dissecting distinct CBL activities. The
results provide guidance to investigate these CBLs in relation to
normal biological functions, functional diversity among individual CBLs
and CBL contributions to anthelmintic-induced intestinal pathology.
Materials--
Fluorogenic peptide substrates included
Ac-YVAD.AMC, Ac-DEVD.AMC, Z-FR.MNA, and Z-RR.MNA. Competitive peptide
inhibitors included Ac-YVAD.CHO and Ac-DEVD.CHO. Irreversible peptide
inhibitors included Z-FA.CHN2, Bt-YVAD.FMK, and Bt-FA.FMK.
All of these substrates were obtained from Enzyme Systems Products
(Dublin, CA).
Parasite and Extracts--
Approximately 10,000 viable L3 larvae
of a Beltsville isolate of H. contortus (21) were used to
orally infect 4-8-month-old parasite-free lambs, which were killed
25-27 days after infection. Adult worms were harvested for dissection
of intestines and for culture to obtain ESP (20).
Protease Assays--
Intestines thawed from pH Profiles--
The pH activity profiles were established using
the following buffers: 100 mM citrate phosphate (pH
3.0-7.0) or 100 mM phosphate (pH 8.0) and 50 mM glycine-NaOH (pH 9.0) containing 10 mM DTT. Classic caspase activity was evaluated under conditions of 10 mM DTT, 312.5 mM HEPES, 31.25% sucrose,
0.3125% CHAPS (pH 7.5). Liberation of the leaving fluorescent groups,
AMC and MNA, was monitored with a fluorescence spectrophotometer (MPF
42A, Perkin Elmer Life Sciences) using excitation and emission
wavelengths of 360 and 460 nm (AMC), respectively, or 340 and 425 nm
(MNA), respectively. Specific activity was determined in picomoles of AMC or MNA liberated per minute per microgram of protein at 37 °C. A
standard curve produced with free AMC or MNA was used for these measurements.
Affinity Isolation of Proteases--
Enzyme reactions were done
by incubating 64 µg of intestinal extract with biotinylated
irreversible substrate inhibitors Bt-YVAD.FMK or Bt-FA.FMK (5 µM) in 0.1 M citrate-phosphate buffer, pH
5.0, containing 10 mM DTT. The reaction mixtures were
incubated for 15 min at 37 °C. Following this incubation, a mixture
of protease inhibitors (100 µM leupeptin, 1 mM pepstatin, 10 mM 1,10-phenanthroline, and 10 µM E-64) was added to the solution. The mixture was
adjusted to 0.2% SDS and incubated for an additional 15 min at room
temperature with gentle agitation. This solution was used for affinity
isolation procedures described below. Inhibition reactions were run by
incubating the intestinal extracts with nonbiotinylated irreversible
inhibitors, Ac-YVAD.FMK (5 µM), Z-FA.CHN2 (5 µM), or E-64 (10 µM) for 5 min (37 °C)
prior to incubation with biotinylated substrate inhibitors.
Proteases conjugated to inhibitors were isolated by incubation with 100 µl of streptavidin-agarose beads (Sigma, St Louis, MO.) for 2 h
at room temperature with gentle rotation. Bound beads were washed 5 times with 0.1 M glycine and 1% SDS (pH 8.0). The streptavidin-agarose beads were boiled in 3X-sample buffer for 15 min.
Eluted proteins were fractionated on 7.5-17.5% gradient SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes as described previously (22). The membranes
were blocked overnight at 4 °C in PBS, Tween 20 (0.1%), and nonfat
dry milk (5%). Membranes were incubated with streptavidin-horseradish
peroxidase conjugate (Kirkegaard & Perry Laboratories, Gaithersburg,
MD) diluted 1:1000 in blocking buffer. Bound streptavidin was
visualized using an enhanced chemiluminescence (ECL) detection system
(Amersham Pharmacia Biotech, Amersham Place, United Kingdom). The
Mr of stained proteins was estimated using
prestained Rainbow molecular mass standards ranging from 14.3 to 200 kDa (Amersham Pharmacia Biotech).
Monoclonal Antibody 42/10.6.1--
This mAb (IgG2a) was used on
immunoblots of affinity isolated proteases to evaluate glycosylation of
CBLs and purity of the preparations. This mAb also provided enhanced
sensitivity for evaluating affinity isolated proteins from ESP.
Immunoblots were done with mAb 42/10.6.1 (2 µg/ml) as described (14).
An isotype control mAb (Babb) against an irrelevant determinant was
used as a control on replicate blots. Bound antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse IgG diluted 1:3000 (Kirkegaard & Perry Laboratories). The blot was developed using ECL.
N-terminal Amino Acid Sequencing--
Proteases were isolated
using Bt-YVAD.FMK (pH 5.0) or Bt-FA.FMK (pH 5.0 and 7.0). Affinity
isolation procedures were optimized in standard assays by titrating the
following components: peptide inhibitors, DTT, streptavidin-agarose
beads, and the incubation time. Affinity isolation assays were then
scaled up 50-fold to produce an estimated 450 pmol of proteins.
Isolated proteases were fractionated on 7.5-17.5% gradient
SDS-polyacrylamide gel electrophoresis and electroblotted onto
polyvinylidene difluoride Immobilon-P membranes (Millipore, Bedford,
MA). Transblotted proteins were visualized by staining with 0.1%
Coomassie Blue for 10 min, followed by rapid destaining. Destained
bands were excised and their N-terminal amino acid sequence determined
using an Applied Biosystems 494/HT Procise sequencing system
(University of Texas Medical Branch, Protein Chemistry Laboratory,
Galveston, TX).
Cytolocalization of H. contortus Intestinal
Proteases--
Location of H. contortus intestinal
proteases was determined using the biotinylated peptide inhibitors
Bt-YVAD.FMK and Bt-FA.FMK. Whole worms were placed in OCT compound
(Tissue-TekR, Miles Inc., Elkhart, IN) and rapidly frozen
in liquid nitrogen. Five micrometer tissue sections were cut using a
cryotome, attached to ProbeOnTM Plus microscope slides (Fisher
Scientific, Pittsburgh, PA), and air-dried for 30 min. Tissue sections
were fixed in 30% methanol, 60% acetone, and 10% distilled
H2O for 30 s at room temperature. Subsequently, the
tissues were rinsed in PBS and incubated at 37 °C for 15 min in 0.1 M citrate-phosphate pH 5.0 buffer, 10 mM DTT,
containing 5 µM Bt-YVAD.FMK or 5 µM
Bt-FA.FMK. Inhibitor control tissue sections were pre-incubated with
nonbiotinylated protease inhibitors or 5 µM E-64 for 15 min at 37 °C, followed by addition of biotinylated inhibitors.
Negative control tissues were incubated without the peptides. The
slides were then washed twice, for 5 min each, in wash buffer and
blocked for 30 min at room temperature using PBS, Tween 20 (0.1%), and
10% normal sheep serum. Endogenous peroxidase inhibitor was included
in reactions (Peroxidase Suppressor, ImmunoPureR; Pierce).
Bound peptides were detected using streptavidin-horseradish peroxidase
conjugate diluted 1:500 in blocking buffer. Streptavidin and biotin
complexes were visualized using the ImmunoPure Metal Enhanced DAB
(3,3'-diaminobenzidine tetrahydrochloride) substrate kit (Pierce).
Peptide Substrate Recognition in H. contortus Intestine and
ESP--
Protease activity was evaluated in intestinal extracts and
ESP using four peptide substrates with the following sequences (designations): FR (cathepsins B/L), RR (cathepsin B), YVAD (caspase 1), and DEVD (caspase 3/CED-3). Initial interest in evaluating caspase
substrates arose from the DNA fragmentation that can be induced by
fenbendazole in intestinal cell nuclei of this parasite (10).
Preliminary experiments at pH 5.0 demonstrated activity against each
substrate, with the exception of DEVD (Fig.
1). When further evaluated, optimal pH
for activity varied according to the substrate used. For example,
activity against the YVAD substrate was restricted predominately to
acidic pH, while activity against the RR substrate was highest around
pH 6.0, and relatively high activity against the FR substrate occurred
from pH 4.5 to 7.5 (Fig. 2,
A-C). No activity against the DEVD substrate was detected under any of the described pH conditions, including preferred caspase
conditions (data not shown). Significant activity against the RR
substrate was not expected in H. contortus intestine based on a previous report (18). In addition, the preferential activity against YVAD under acidic conditions is inconsistent for caspases, which have neutral pH requirements (11).
Inhibitory effects of leupeptin and E-64 indicated that the majority of
the activities against FR, RR, and YVAD substrates from intestine and
ESP are due to cysteine proteases (Table
I). Some variation on inhibition was
observed among substrates with iodoacetic acid. Similar inhibitory
results were obtained for FR and RR substrates at pH 5.0-6.0 (data not
shown). Although 1,10-phenanthroline had moderate inhibitory effect on
activities (57-87%), cysteine protease inhibition by
1,10-phenanthroline may be ascribed to a possible metal ion chelate.
Although the relatively low inhibitory effect of EDTA supports this
possibility, activity contributed by noncysteine proteases cannot be
completely ruled out by these experiments. In contrast to the relative
insensitivity of caspases to E-64 and leupeptin, intestinal activity
against the YVAD substrate was ablated by both inhibitors. This result and observations on pH requirements suggested that activity against the
YVAD substrate is conferred by noncaspase cysteine protease activity.
Cross-inhibition of Protease Activity Using Peptide
Substrates--
The possibility that related cysteine proteases confer
activity against each of the substrates was evaluated by
cross-inhibition experiments. Fluorogenic peptide substrates and
peptide-based irreversible inhibitors were used in these experiments.
Since no inhibitor construct was available for the RR substrate,
experiments focused on the YVAD- and FR-based inhibitors. Additionally,
a FA substrate modified by diazomethane (CHN2) was
previously shown to inhibit H. contortus cysteine protease
activity (20) and was used here. Activity against each substrate was
inhibited at pH 5.0 by the irreversible inhibitors Ac-YVAD.FMK and
Z-FA.CHN2 (Fig. 3). The
inhibitor Ac-YVAD.CHO had similar effects on both YVAD and FR
hydrolysis (data not shown). The high level of cross-inhibition suggested that a major portion of the activity against all substrates resided in the same enzymes at pH 5.0.
In contrast to YVAD, hydrolysis of the FR substrate was relatively high
at pH 7.0. However, this activity was inhibited by Z-FA.CHN2 (92%), Ac-YVAD.FMK (94%), and Ac-YVAD.CHO
(78%). This result suggests that pH 7.0 inhibited protease binding by
YVAD to a much lesser extent than protease hydrolysis of this
substrate, which will be discussed below. Nevertheless, the higher
level of inhibition by the irreversible inhibitor Ac-YVAD.FMK compared with Ac-YVAD.CHO may indicate low level hydrolysis of this substrate at
pH 7.0.
Affinity Isolation of Cysteine Proteases--
Biotinylated peptide
substrates modified by a fluoromethylketone reactive group were
used to isolate, characterize, and identify proteases that hydrolyze
the peptide substrates described. Use of Bt-YVAD.FMK led to isolation
of multiple prominent bands ranging from 29 to 37 kDa (Fig.
4A), with a 42-kDa band that
was weakly visible (lane 2). A 33-kDa band was
most prominent in multiple experiments. A similar profile of proteins
was isolated by Bt-FA.FMK (lane 6), although the
signal was always reduced compared with Bt-YVAD.FMK. A 33-kDa band was
also most prominent in this lane, followed by a 37-kDa band. E-64
eliminated bands isolated by each of the biotinylated substrates,
indicating they are cysteine proteases. Use of the nonbiotinylated
inhibitor Ac-YVAD.FMK produced similar results. A Bt-DEVD.FMK probe
failed to isolate any detectable bands from intestinal lysates (data
not shown). The diazomethane inhibitor Z-FA.CHN2 was more
discriminating, causing obvious reduction of only the 33-kDa band in
preparations isolated by both the Bt-YVAD.FMK and Bt-FA.FMK. The two
discernible weaker bands remaining at this position may reflect
incomplete inhibition. Since Z-FA.CHN2 reduced activity
against the FR and YVAD fluorogenic substrates by 100% and 94%,
respectively (Fig. 3), proteases from the 33-kDa band may be largely
responsible for hydrolyzing these substrates. Alternatively, Z-FA.CHN2 could function in fluorogenic assays as a
competitive inhibitor against proteases that are otherwise resistant to
irreversible binding by this inhibitor.
Monoclonal Antibody 42/10.6.1--
Previous results suggested that
CBLs are modified by the periodate-sensitive determinant recognized by
mAb 42/10.6.1 (16). This determinant occurs on a multitude of
membrane/secreted/excreted proteins from H. contortus (14).
Immunoblot analysis showed that most, if not all, proteins
affinity-purified by Bt-YVAD.FMK and Bt-FA.FMK are modified by the mAb
42/10.6.1 determinant. In addition, the restricted size range of
isolated proteins, compared with whole intestinal lysates, demonstrated
a high level of purity in these preparations (Fig. 4B).
Substrate and pH-dependent Intestinal Protease
Isolation--
pH effects were evaluated on affinity isolation at pH
5.0 and 7.0. The bands isolated by Bt-YVAD.FMK at pH 5.0 were
eliminated at pH 7.0 (Fig. 5). In
contrast, a similar profile of bands was isolated by Bt-FA.FMK
regardless of the pH conditions used. These results are consistent with
the pH effects on hydrolysis of fluorogenic substrates.
Comparison of Affinity Isolated ESP and Intestinal
Proteins--
Characteristics established for presumptive intestinal
proteases were next applied to evaluate proteins in ESP.
Streptavidin-horseradish peroxidase lacked necessary sensitivity for
this purpose. Alternatively, mAb 42/10.6.1 provided for a sensitive
detection method. A size range of proteins was isolated from ESP by
each substrate that paralleled those found in intestine (Fig. 5). The
proteins isolated behaved in a substrate- and pH-dependent
manner that paralleled intestinal proteins. The results also establish
that the isolated proteins are modified by the mAb determinant.
Collectively, results agree with a previous report (18) that the ESP
proteins most likely represent intestinal cysteine proteases excreted
from the parasite.
Identification of CBLs from Affinity Isolated
Proteins--
N-terminal sequencing was done on two isolated
intestinal protein bands. The 33-kDa band was chosen based on evidence
of activity against both YVAD and FR substrates. The 37-kDa band was
chosen due to the relative resolution of this band and the resistance of the band to inhibition by Z-FA.CHN2. N-terminal
sequences were obtained for bands isolated at pH 5.0 (FA, YVAD) and 7.0 (FA). Current data bases contain an estimated 11 published H. contortus CBL sequences (25) and expressed sequence tags for an
additional 11 unique CBLs.2
The N-terminal sequences from each band had greatest similarity with
H. contortus CBLs (Fig. 6).
The sequences began at a uniform site consistent for pro-region
cleavage, which is expected for the active proteases. Each sequence
(20-24 residues) of the 37-kDa protein band isolated by each
inhibitor, regardless of pH, exactly matched the H. contortus CBL AC-4 (Fig. 6). The residue corresponding to a single
predicted cysteine residue was ambiguous in the protein sequences. The
AC-4 sequence has multiple amino acids that are distinct from other
CBLs, especially from consensus residues 11-17, providing a high
degree of confidence in this identification.
Of the sequences from the 33-kDa band (10-17 residues), three exactly
matched predicted amino acid sequence of the H. contortus CBL GCP-7 and an EST sequence (Hcgls1H4), which appears to represent GCP-7.2 These sequences were obtained from bands isolated
by both the FA and YVAD affinity probes. The 33-kDa band obtained by FA
isolation at pH 7.0 produced two alternative sequences due to
heterogeneity (D/E) at the first residue (see fa7a and fa7b). The
heterogeneity is likely to reflect more than one protease in this band.
However, the dominant sequence clearly matches GCP-7 and the sequence
is of sufficient length to instill a high degree of confidence in this identification.
Cytolocalization of Intestinal Proteases--
Cytolocalization of
the intestinal CBLs identified was determined with the biotinylated
peptide inhibitors. In parallel with affinity isolation results,
Bt-YVAD.FMK proved more sensitive in histological assays than Bt-FA.FMK
(Table II). In sections of whole worms,
Bt-YVAD.FMK binding was found only at the microvillar surface of the
worm intestine (Fig. 7A). E-64
completely inhibited substrate binding to microvilli (Fig.
7B). Some background peroxidase activity localized to
intestinal cytoplasm (Fig. 7C), but did not extend to
microvilli. In other experiments, Z-FA.CHN2 and Ac-YVAD.FMK
completely inhibited binding of the biotinylated peptides to intestinal
proteases in the H. contortus intestines (Table II).
Experiments with Bt-FA.FMK produced weaker staining on microvilli that
could be inhibited by E-64 and Z-FA.CHN2 (data not shown). From these results, we conclude that active intestinal CBLs localize to
gut microvilli in vivo.
Our results extend previous observations in which intestinal
cysteine protease activity of H. contortus was shown to be
capable of hydrolyzing a FR substrate (18). Those experiments also used affinity labeling with Bt-FA.FMK (18) to identify four protein bands
(30, 34, 37, and 41 kDa) from ESP. We isolated intestinal proteases of
similar size with the same reagent and showed that two of these
proteins are CBLs with identity to H. contortus GCP-7 (33 kDa) and AC-4 (37 kDa). Specificity of these CBLs was extended to
include the YVAD substrate. Although each band may contain multiple
proteases, our results provide confidence in identification of the
predominant CBLs in these bands. We anticipate that proteins isolated
in other bands also represent distinct CBLs.
In contrast to previous results (18), significant activity was detected
against a cathepsin B substrate (RR) that was inhibited by FA and YVAD
inhibitor analogues. The result suggests that CBLs are responsible for
this activity, also. Reasons for the discrepancy are unclear, since
similar substrates and conditions were employed in both investigations.
Our result is curious, since many H. contortus CBL sequences
lack a glutamate corresponding to position 245 in cathepsin B, which is
required for similar activity in cathepsin B (16, 24). However, the CBL
HmCP6 has a glutamate at this position (7) and other unidentified CBLs
might also. Further, we cannot exclude that different laboratory
isolates of H. contortus vary in repertoire of CBLs and,
hence, substrates recognized. Resolving these issues should be
facilitated with the established connection between CBLs and intestinal
cysteine protease activity in H. contortus.
Each of the CBLs identified exhibited diverse properties. Both the
GCP-7 and AC-4 proteases bound to the FA and YVAD affinity probes under
conditions of pH 5.0, demonstrating diverse substrate specificity.
Likewise, both bound to the FA affinity probe at pH 7.0, demonstrating
broad pH requirements. Other unidentified proteins that represent
presumptive CBLs behaved similarly. Therefore, individual CBLs appear
to possess labile properties that support activity against diverse
substrates under a variety of pH conditions. Genes for the two CBLs
identified (GCP-7 and AC-4) have been cloned (1, 15) and offer a means
to investigate protease characteristics that determine these properties.
In contrast, failure to hydrolyze the DEVD substrate indicates one
limit to CBL activity. In addition, pH requirements were more
restricted for hydrolysis of the YVAD and RR substrates. For YVAD,
inhibition of hydrolysis at pH 7.0 is not readily explained by
inhibition of substrate binding, since YVAD substrate inhibitors abrogated activity against FR substrates at this pH. Therefore, inhibition of actual hydrolysis, not substrate binding, appears to
explain these observations. Furthermore, CBLs isolated by both Bt-FA.FMK and Bt-YVAD.FMK appeared to be the same. This result indicates that hydrolysis of the YVAD substrate at pH 7.0 was inhibited
in CBLs that otherwise had catalytic activity against the FR substrate.
Therefore, this effect may reflect displacement of the YVAD substrate
in the binding pocket due to a pH influence on the CBL or substrate.
For instance, a pH-dependent specificity switch occurs in
cruzain, a Trypanosoma cruzi cathepsin L-like enzyme that can bind both hydrophobic and basic residues at different pH environments (27). Insufficient information is currently available
for comments regarding the protease. However, the P1 aspartate in YVAD
is the most likely substrate residue to be altered in charge at pH 7.0, which may account for the inhibition observed. Caspase substrate
inhibitors YVAD.FMK and VAD.FMK have been shown to inhibit cathepsin B
(26), but parallel observations on pH-dependent substrate
binding were not reported for this protease. Our results raise the
possibility that protonation of the P1 aspartate is important for
caspase substrate interactions with cathepsin B.
Regarding the significance of YVAD substrate hydrolysis, it may be
important that acidic residues (aspartate or glutamate) immediately
precede the predicted proregion cleavage site for HmCP6, AC-3, and
AC-4. This position would represent the P1 site for CBLs. Since
cathepsin B proregion cleavage is autocatalytic (28, 29), there may be
a biological need for at least a subset of CBLs to hydrolyze substrates
with P1 acidic residues.
In contrast to fluoromethyl ketone inhibitors, the diazomethane
derivative of the FA substrate preferentially inhibited affinity isolation of the GCP-7 protease band. The fluoromethylketone derivative is more specific to active site cysteines than diazomethane (27). However, this property alone should not discriminate among individual proteases, whereas structural differences among CBLs might. CBL sequence diversity in nematodes has stimulated much speculation regarding the functional significance of this diversity (3, 4, 11, 16).
The preferential inhibition of the GCP-7 band provides the first direct
evidence of functional differences among H. contortus CBLs.
AC-4 and GCP-7 sequences are quite divergent and were associated with
distinct CBL clades from H. contortus (16). Notable
differences in predicted S'2 binding sites distinguished members of
these clades (16). In this context, differences such as that indicated
by Z-FA.CHN2 are not unexpected. This discriminating property of the diazomethane inhibitor might have value for
characterizing recombinant CBLs and dissecting CBL properties in
vivo. Alternatively, this inhibitor would appear to have
limitations for use as a general irreversible inhibitor of CBL
functions in vivo.
One objective of the research was to establish definitions and
approaches that would support research on CBLs in a biological context.
For instance, CBLs have been immunolocalized to H. contortus microvilli (7, 8). Each of the affinity isolated CBLs evaluated had N
termini consistent with pro-region cleavage and protease activation.
Hence, CBLs detected on microvilli by affinity probes are most likely
active enzymes. These and other results (7, 8, 18) support that CBLs
have a major role in nutrient digestion by H. contortus.
Further, 17% of expressed sequence tags from H. contortus
intestine were cbl genes, which dominated all other intestinal genes/gene families found.2 These results
indicate a prominent role for CBLs in the biology of H. contortus. In addition, fenbendazole treatment cause secreted intestinal proteins modified by the mAb 42/10.6.1 determinant to become
dispersed in the cytoplasm of H. contortus intestinal cells
that undergo degeneration (10). The dispersed proteins are likely to
include CBLs, since CBLs are expressed in the intestine (7, 15, 16),
are transported to microvilli (10), and, as shown here, are modified by
the mAb 42/10.6.1 determinant. With the broad pH requirements observed,
CBLs become potential candidates for mediating intestinal pathology
induced by the anthelmintic fenbendazole. Finally, CBL activity can be
distinguished from classic caspase activity, which may be important for
elucidating cellular mechanisms of fenbendazole-induced DNA
fragmentation (10).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C were
homogenized in PBS containing 1% Triton X-100. Protein concentration
of lysates was determined by using the bicinchoninic acid protein assay
reagent (Pierce). The extracts were stored in 200-µl aliquots at
80 °C. Protease activity was initially determined using the
fluorogenic di- and tetrapeptide substrates, Z-FR.MNA, Z-RR.MNA,
Ac-YVAD.AMC, and Ac-DEVD.AMC at 50 µM concentrations.
Reactions were performed in 1.5-ml volumes with 10 µg of intestinal
extracts or ESP and incubated at 37 °C for 2 h. Samples were
evaluated at 30-min intervals for 2 h and activity reported for
the 2-h incubations. Worm samples without substrates served as negative
control. Inhibition studies incorporated protease-specific class
inhibitors including 1 mM phenylmethylsulfonyl fluoride, 10 µM E-64, 100 µM leupeptin, 1 µM pepstatin, 100 µM iodoacetic acid, 10 mM 1,10-phenanthroline, or 5 mM EDTA. In
addition, substrate-based dipeptide (Z-FA.CHN2) and
tetrapeptide (Ac-YVAD.CHO, Ac-YVAD.FMK) protease inhibitors were used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Protease activity in the intestines and ESP
of adult H. contortus. Intestinal extracts (int)
and ESP (10 µg) were assayed at pH 5.0 at 37 °C for 2 h.
Fluorogenic substrates Z-FR.MNA, Z-RR-MNA, Ac-YVAD.AMC, and Ac-DEVD.AMC
were used at 50 µM. Enzyme activity was expressed as
picomoles of AMC/min/µg or picomoles of MNA/min/µg, respectively.
Means of triplicate assays are presented, and bars represent
standard deviations.
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Fig. 2.
pH effects on intestinal and ESP protease
activity. Protease activities in intestinal extracts
(int) and ESP of H. contortus were assayed at pH
3.0-9.0 with Z-FR.MNA (A), Z-RR.MNA (B), and
Ac-YVAD.AMC (C). Means of triplicate assays are shown.
Bars represent standard deviations.
Mean percentage of inhibition of protease activity from extracts of
adult H. contortus intestine
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Fig. 3.
Substrate-based cross-inhibition of
intestinal protease activities. Intestinal extracts (10 µg) were
pre-incubated for 5 min with peptide substrate inhibitors
Ac-YVAD.FMK or Z-FA.CHN2 (50 µM) at pH 5.0 and then incubated with fluorogenic substrates Ac-YVAD.AMC, Z-FR.MNA,
or Z-RR.MNA at 37 °C for 2 h. Results were expressed as mean
percentage of inhibition of activity compared with reactions with no
inhibitors. Bars represent standard deviations of triplicate
assays.
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Fig. 4.
Affinity isolation of H. contortus
intestinal proteases with biotinylated peptide inhibitors.
A, intestinal lysates (64 µg) were incubated without
Bt-YVAD.FMK (lane 1), with Bt-YVAD.FMK
(lanes 2-5), or with Bt-FA.FMK (lanes
6-9) at pH 5.0, affinity isolated, electrophoretically
fractionated, and detected (see "Experimental Procedures"). Samples
for competition inhibition assays were pre-incubated with
nonbiotinylated peptide inhibitors identified below the
panel. Arrows indicate two prominent bands identified at 33 and 37 kDa. B, immunoblot of intestinal proteins purified
with Bt-YVAD.FMK (lane 1), purified with
Bt-FA.FMK (lane 2), or from whole worm extracts
(lane 3), using mAb 42/10.6.1 (2 µg/ml).
Isolated proteins from 64 µg of extracts or 25 µg of whole
intestine were used. No reactivity was observed on a replicate blot
using an isotype control mAb (IgG2a). Arrows point to the
33- and 37-kDa proteases.
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Fig. 5.
Differential pH effects on substrate
binding. Intestinal lysates and ESP were reacted with Bt-YVAD.FMK
(B-Y-F) at pH 5.0 or pH 7.0, or Bt-FA.FMK (B-F-F)
as indicated. Isolated proteases were then detected with
streptavidin-horseradish peroxidase (intestines) or by immunoblot
methods with mAb 42/10.6.1 (ESP) as described for Fig.
4B.
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Fig. 6.
Identification of affinity isolated
proteases. N-terminal amino acid sequences (in bold)
were obtained for affinity isolated protein bands (see "Experimental
Procedures") and aligned with previously reported H. contortus cathepsin B-like cysteine protease sequences. Proteases
were isolated with Bt-YVAD.FMK (yv) or Bt-FA.FMK
(fa) at pH 5.0 (5) or pH 7.0 (7). The
33-kDa protease sequences were identical to GCP-7
(underlined) and the 37-kDa protease sequences were
identical to AC-4 (underlined). fa7a and
fa7b refer to a degenerate residue at the first position
(D/E) for the 33-kDa band isolated by Bt-FA.FMK at pH 7.0. "X" represents an ambiguous amino acid predicted to be a
cysteine residue in the sequences. P/A indicates the
expected junction of the propeptide (P) cleavage site and
active enzyme (A). Vertical bars at
the bottom of the figure show the position of the AC-4 amino
acids that are distinct from other CBLs. Numbers in
parentheses refer to GenBank accession numbers:
1, A172357; 2, A1723422; 3, A11723514;
4, A1723554; 5, A1723598; 6, A1723446;
7, A1723444; 8, A1723442; 9, A1723407;
10, Z81327; 11, Z69346; 12, Z69344;
13, Z69345; 14, A1723403; 15,
A1723612; 16, AF046229; 17, A1723380;
18, A1723416; 19, Z69342; 20, Z69343;
21, A1723392; 22, A1723405; 23,
C48435; 24, M31112; 25, M34860; 26,
D48435; 27, B48435.
Binding of di and tetrapeptide substrate probes to microvilli of H. contortus intestine at pH 5.0
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Fig. 7.
Cytolocalization of H. contortus
intestinal CBLs. Sections from frozen whole worms were
incubated with Bt-YVAD.FMK (A, 5 µM), E-64 (5 µM) followed by Bt-YVAD.FMK (B), or no
peptides (C). Biotinylated peptide binding was detected
using streptavidin-conjugated horseradish peroxidase. White
horizontal arrow, microvilli; black
vertical arrow, reproductive organs;
black horizontal arrows, body wall
musculature.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We appreciate the excellent technical support of Xiaoya Cheng and Stewart G. Bohnet in this work. Protein sequencing was done with support from the Protein Chemistry Laboratory at the University of Texas Medical Branch Educational Cancer Center (Galveston, TX) and the University of Texas Medical Branch NIEHS center grant.
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FOOTNOTES |
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* This work was supported by United States Department of Agriculture Grant NRICGP 98-35204-6461.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 509-335-6320;
Fax: 509-335-8529; E-mail: shompole@vetmed.wsu.edu.
Published, JBC Papers in Press, October 13, 2000, DOI 10.1074/jbc.M007321200
2 S. Shompole and D. P. Jasmer, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: cbl and CBL, cathepsin B-like cysteine protease gene and protein, respectively; AMC, 7-amino-4-methylcoumarin; Ac-YVAD, acetyl-Tyr-Val-Ala-Asp; Ac-DEVD, acetyl-Asp-Glu-Val-Asp; CHO, aldehyde; MNA, 4-methoxy-2-naphthalamine; Z-FR, benzyloxycarbonyl-Phe-Arg; Z-RR, benzyloxycarbonyl-Arg-Arg; CHN2, diazomethane; Bt, biotin; FMK, fluoromethyl ketone; E-64, trans-epoxysuccinyl-L-leucylamide-(4-guanidino)-butane; ESP, excretory and secretory products; DTT, dithiothreitol; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mAb, monoclonal antibody.
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REFERENCES |
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