From the
Parasitology and
Physical Biochemistry, National Institute for
Medical Research, Mill Hill, London NW7 1AA, United Kingdom
Received for publication, April 11, 2003 , and in revised form, May 20, 2003.
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ABSTRACT |
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INTRODUCTION |
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Subtilases are synthesized as zymogens, which consist minimally of a signal peptide, a propeptide domain, and a catalytic domain. The propeptide functions as an intramolecular chaperone, being essential for correct folding of the catalytic domain and for enzyme maturation (13). Folding is generally rapidly followed by autoproteolytic cleavage at the junction between the propeptide and mature domain. The cleaved propeptide often remains transiently associated with the catalytic domain in a noncovalent complex. In many cases, subtilase propeptides are potent inhibitors of their cognate catalytic domains, with inhibition constants in the nanomolar range (1418), and so eventual enzyme activation generally requires further proteolytic digestion and release of the propeptide (14, 15). PfSUB-1 maturation during secretory transport in the parasite involves two sequential autocatalytic processing events that convert the precursor first to a p54 form by cleavage of the propeptide and subsequently to a p47 form by further cleavage within the N-terminal segment of p54 (11). Interestingly, when produced in a baculovirus expression system, recombinant PfSUB-1 (rPfSUB-1) is mostly secreted in the p54 form, a substantial fraction of which is noncovalently linked to its cognate propeptide, p31 (11, 12).
To better understand the activation of PfSUB-1, we have examined the role of the propeptide and explored the possibility that it can act as a specific inhibitor. After recombinant expression and characterization of the propeptide, we have investigated its structure, its interactions with both baculovirus-derived and native enzymes, and the specificity and stability of these interactions. Analyses of various mutants of the propeptide have indicated the regions and residues that determine its interactions with the catalytic domain.
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EXPERIMENTAL PROCEDURES |
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Site-directed MutagenesisThe desired mutations within the PfSUB-1 propeptide were introduced by PCR-mediated site-directed mutagenesis using the F1 forward primer in combination with the reverse primers indicated below. Three different rp31 mutants (called rp31-VSAL, rp31-ASAD and rp31-ASAL) were generated. In the rp31-VSAL mutant, the C-terminal residue Asp219 was mutated to Leu by replacement of the terminal CTA codon with CTT using the reverse oligonucleotide 5'-AGAGGAGAGTTAGAGCCTTAAAGAGCGGACACCAACTTGTC-3' (the mismatched codon is underlined). In the rp31-ASAD mutant, Val216 was similarly mutated to Ala using the reverse oligonucleotide 5'-AGAGGAGAGTTAGAGCCTTAATCAGCGGAAGCCAACTTGTCAGAC-3'. For the rp31-ASAL double mutant, Asp219 was mutated to Leu and Val216 was replaced with Ala using the reverse oligonucleotide5'-AGAGGAGAGTTAGAGCCTTAAAGAGCGGAAGCCAACTTGTCAGAC-3'. DNA fragments encoding the mutants were cloned into pET-30Xa/LIC, and the mutations were verified on both strands by sequencing as described above.
Protein Expression and Purification and Antiserum
Production rp31 and mutant rp31 sequences were expressed in E.
coli BL21-Gold (DE3) pLysS (Stratagene). One liter of LB medium
containing 30 µg/ml kanamycin was inoculated with 4 ml of fresh overnight
culture and grown at 37 °C to A600 = 0.5 prior to
induction with 1 mM
isopropyl--D-thiogalactopyranoside. After 4 h of induction,
the cells were pelleted, resuspended in 50 ml of 50 mM phosphate
buffer (pH 8.0) containing 300 mM NaCl, and lysed by sonication,
and the insoluble material was removed by centrifugation. The resulting
supernatant was supplemented with 1 mM phenylmethylsulfonyl
fluoride, 1 µM pepstatin, 100 µM leupeptin, and
0.3 µM aprotinin and clarified by passage through a 0.22-µm
filter.
Purification of all of the recombinant propeptide constructs to homogeneity
was by the same three-step procedure. To the 50-ml supernatant was added
40 ml of nickel-nitrilotriacetic acid-agarose (QIAGEN Inc.). The gel
suspension was mixed on ice for 30 min, transferred to a sintered glass
funnel, washed with 5 gel volumes of 2 mM imidazole in 50
mM phosphate buffer (pH 8.0) containing 300 mM NaCl, and
eluted with 2 gel volumes of 150 mM imidazole in the same buffer.
Eluted proteins were concentrated by ultrafiltration at 4 °C with a YM-10
membrane (Amicon, Inc.) and then chromatographed on a Superdex 200 26/60
prep-grade column (Amersham Biosciences) equilibrated in 20 mM
Tris-HCl (pH 8.2) containing 150 mM NaCl. The propeptide peak was
detected by SDS-PAGE (19) and
staining with Coomassie Blue R-250. Pooled peak fractions were loaded onto a
1-ml RESOURCE Q column (Amersham Biosciences) pre-equilibrated in 20
mM Tris-HCl (pH 8.2) and 150 mM NaCl. The column was
washed with the same buffer and then eluted with a linear gradient of NaCl
(150600 mM) in the same buffer. The propeptide peak was
pooled and stored in aliquots at 70 °C. Electrospray mass
spectrometric analysis of the purified protein was performed as described
previously (11). A mouse
polyclonal antiserum was raised against the purified protein using standard
procedures. Expression of baculovirus-derived rPfSUB-1 in insect cells and
protease purification were as described previously
(12).
Cleavage of the His6 and S Tags from
rp31The N-terminal fusion peptide containing the His6
and S tags was removed from rp31 using Factor Xa (Roche Applied Science). A
solution of purified rp31 was adjusted to 50 mM Tris-HCl (pH 8.2),
100 mM NaCl, and 1 mM CaCl2; Factor Xa was
added to obtain a protease/substrate ratio of 1:56 (w/w); and digestion was
allowed to proceed at room temperature for 4 h. Protease activity was stopped
by the addition of 1 mM phenylmethylsulfonyl fluoride. An equal
volume of nickel-nitrilotriacetic acid-agarose was added to the digest, and
unbound material containing cleaved rp31 (rp31SH) was recovered,
supplemented with 10 mM EGTA, and purified by gel filtration as
described above. The rp31
SH peak was concentrated by ultrafiltration at
4 °C using a YM-3 membrane (Amicon, Inc.) and stored at 70
°C.
Determination of Protein Purity and ConcentrationThe amino acid composition and concentration of purified rp31 were determined in duplicate amino acid analyses by Dr. Peter Sharratt (Protein and Nucleic Acid Chemistry Facility, Cambridge University, Cambridge, UK). The concentrations of the various purified rp31 mutants were then determined by a comparative reversed-phase high pressure liquid chromatography (RP-HPLC) method. Samples of purified rp31 and its mutant forms were chromatographed on a Vydac C18 RP-HPLC column (4.6 mm x 25 cm) and eluted with a 927% (v/v) gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid, with absorbance monitored at 215 nm. Each recombinant protein eluted as a single peak under these conditions. The concentration of each mutant was extrapolated by comparing its peak height upon RP-HPLC with the peak height of a known amount of rp31.
CD MeasurementsCD spectra of rp31 and rp31SH were
recorded from 260 to 195 nm (or from 260 to 210 nm in the presence of urea) at
20 °C in a fused silica cuvette of 1-mm path length using a Jasco J-715
spectropolarimeter. Protein concentrations were
0.1 mg/ml, and the buffer
was 5 mM Tris-HCl (pH 8.2) and 67.5 mM NaCl with or
without 5 M urea (for rp31) or 16.6 mM Tris-HCl (pH 8.2)
and 125 mM NaCl (for rp31
SH). The spectra are the averages
of multiple scans recorded using a scan speed of 200 nm/min and a response
time of 0.25 s. Appropriate buffer blanks were subtracted from all spectra.
For deconvolution, spectra were subjected to multicomponent secondary
structure analysis using the SELCON3 algorithm
(20).
PfSUB-1 Enzyme Activity and Inhibition AssaysKinetic
experiments assessing the effect of recombinant propeptide constructs on the
activity of baculovirus-derived rPfSUB-1 were as described previously, based
on the cleavage of the tetramethylrhodamine-labeled fluorogenic peptide
substrate pepF1-6R (12,
21). Briefly, 90 µl of
pepF1-6R (2 µM final concentration) in 50 mM Tris-HCl
(pH 8.2), 12 mM CaCl2, and 0.05% (w/v) Nonidet P-40 was
added to a 3 x 3-mm path length cell. The solution was allowed to
stabilize to 37 °C in the spectrofluorometer for 2 min before the addition
of 10 µl of purified rPfSUB-1 (74 nM final concentration).
Increase in fluorescence, reflecting cleavage of pepF1-6R, was measured over
the ensuing 4 min. To determine the inhibitory activity of the recombinant
propeptides, 1 µl of different concentrations of the recombinant
propeptides or 2 µl of an inhibitor control (3,4-dichloroisocoumarin at 80
µM final concentration) was added directly to the reaction with
rapid mixing, and the hydrolysis rate was measured for an additional 5 min.
All experiments were performed in triplicate and used concentrations of
pepF1-6R well below its Km, under which
conditions, Ki(app)
Ki
(22). In the absence of
inhibitors, the rate of hydrolysis over this period was linear, and
10%
of the total substrate was hydrolyzed. Following each assay, the cell was
thoroughly cleaned by treatment with chromic acid.
P. falciparum schizont extracts containing endogenous PfSUB-1 were prepared by successive solubilizations with Triton X-100 and CHAPS as described previously (11, 12). To determine inhibition of native PfSUB-1 by rp31, the schizont extract was incubated with an unlabeled synthetic peptide substrate called PEP1 (2 mM final concentration) in the absence or presence of purified rp31 (84 nM final concentration) for 4 h at 37 °C, and then cleavage of PEP1 was assessed by RP-HPLC as described previously (12).
Selectivity of Inhibition by rp31The selectivity of rp31 as a subtilase inhibitor was assessed by measuring its capacity to inhibit the activity of subtilisin BPN' (Sigma P-4789) or subtilisin Carlsberg (Sigma P-5380). The assay was based on the hydrolysis of N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA) by these proteases, essentially as described by DelMar et al. (23), in 50 mM Tris-HCl (pH 8.2), 12 mM CaCl2, and 0.05% (w/v) Nonidet P-40 at room temperature. Kinetic measurements were performed using a Unicam UV1 spectrofluorometer, measuring absorbance at 412 nm. For Km determination, concentrations of Suc-AAPF-pNA between 60 µM and1mM were used to determine initial hydrolysis rates, which were plotted against substrate concentration, fitting the data to the hyperbolic Michaelis-Menten rate equation by nonlinear regression using the program GraFit 5 (Version 5.0.4, Erithacus Software Ltd., Horley, UK) (24). For inhibition assays, 500 µl of the substrate at 60 µM in a 10-mm path length cell was supplemented with 2.5 µl of subtilisin BPN' or subtilisin Carlsberg (25 nM final concentration). Increase in absorbance was measured over the ensuing 15 min to obtain an initial hydrolysis rate, before direct addition to the mixture of various concentrations of purified rp31 (16.8, 42, 84, and 168 nM final concentrations) or phenylmethylsulfonyl fluoride (0.5 mM final concentration) as an inhibitor control. The subsequent hydrolysis rate was measured for an additional 10 min. Note that, in the absence of inhibitors, the rate of hydrolysis was linear over the entire period of measurement, and no more than 10% of the total available substrate was hydrolyzed.
Stability of rp31 and rp31 MutantsThe stability of rp31 and its various mutants to digestion by rPfSUB-1 in the presence of the substrate pepF1-6R was assessed by SDS-PAGE and Western blotting. To 90 µl of 2 µM pepF1-6R in 50 mM Tris-HCl (pH 8.2), 12 mM CaCl2, and 0.05% (w/v) Nonidet P-40 were added 10 µl of purified rPfSUB-1 (61.5 nM final concentration) and 1 µl of recombinant propeptide at various concentrations. The mixture was incubated at 37 °C. At intervals, aliquots were removed, and protease activity was terminated by boiling in Laemmli dissociation buffer containing 100 mM dithiothreitol. All samples were then subjected to SDS-PAGE and electroblotted onto Hy-bond-C membrane (Amersham Biosciences). The membranes were blocked by incubation in 5% (w/v) nonfat milk in phosphate-buffered saline (PBS) and incubated with mouse anti-rp31 antiserum, followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG antibody. Antigen-antibody interactions were visualized by enhanced chemiluminescence (SuperSignal West Pico chemiluminescent substrate, Pierce).
Binding of rPfSUB-1 to rp31 and rp31 MutantsRecombinant
propeptide constructs, along with molecular mass marker proteins (Amersham
Biosciences) as controls for nonspecific binding of PfSUB-1, were subjected to
SDS-PAGE, and the proteins were either stained with Coomassie Blue R-250 or
transferred to Hybond-C membrane. The membranes were blocked by incubation in
5% (w/v) nonfat milk in PBS and then incubated for 1 h at room temperature in
PBS containing purified rPfSUB-1 (3 nM final concentration)
before washing and probing with a rabbit antiserum specific for the catalytic
domain of PfSUB-1 (8), followed
by horseradish peroxidase-conjugated anti-rabbit IgG antibody.
Antigen-antibody interactions were visualized by enhanced
chemiluminescence.
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RESULTS |
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A sample of purified rp31 was treated with Factor Xa to remove the
N-terminal His6 and S tags, and the resulting protein (called
rp31SH) was analyzed by CD under nondenaturing conditions in parallel
with intact rp31 (Fig.
1C). The secondary structure of rp31
SH measured
from the CD spectrum was calculated to be 33.4% unordered. The spectrum also
displayed some negative ellipticity at 208 and 222 nm, indicating the presence
of secondary structure that, upon deconvolution, could be attributed to 19.5%
-helix and 26.3%
-sheet (Fig.
1C). It is worth noting that, as mentioned above, the
PfSUB-1 propeptide sequence is not rich in aromatic residues, side chains of
which can contribute to the CD spectrum and provide potentially misleading
data (25). Our results
indicate that the recombinant propeptide adopts significant secondary
structure in isolation, without the presence of the cognate PfSUB-1 catalytic
domain. Under similar conditions, the CD spectrum of intact rp31 revealed an
only slightly lower degree of secondary structure, consisting of 13.8%
-helix and 21.9%
-sheet. Importantly, this could be removed by
denaturation with 5 M urea (Fig.
1C). Our data indicate that the N-terminal sequence
comprising the His6 and S tags does not contribute to the secondary
structure of the protein and, more importantly, does not prevent folding of
its propeptide component. This result validated the use of rp31 for the
additional studies described below.
Inhibitory Effect of rp31 on Recombinant and Authentic PfSUB-1The ability of rp31 to inhibit the proteolytic activity of rPfSUB-1 was determined in kinetic assays using the fluorescent substrate pepF1-6R. Preliminary experiments indicated that the addition of a molar excess of rp31 to an ongoing hydrolysis reaction resulted in immediate conversion of the linear progress curve to a less steep steady-state linear curve, with no evidence of a hyperbolic transition period, suggesting that the propeptide can act as a typical rapid equilibrium competitive inhibitor. Additional experiments showed that this effect was reproducible and concentration-dependent (Fig. 2A). The apparent tight binding inhibition constant (Ki(app)) of the interaction was calculated using the method of Nicklin and Barrett (22), which uses the slope of the progress curve in the absence of the inhibitor under test (the initial velocity, vo) and the slope of the progress curve immediately following the addition of inhibitor (the inhibition velocity, vi). A plot of (vo/vi)1 versus inhibitor concentration has a slope of 1/Ki(app) (Fig. 2B). Using this method, the experimentally determined Ki(app) was 5.3 ± 0.4 nM (Table I). At the substrate concentration used, which is substantially below its Km, this is unlikely to be significantly different from the Ki (21, 22). Another E. coli derived recombinant fusion protein possessing the same N-terminal fusion partner as rp31, but containing an irrelevant malarial protein sequence, displayed no detectable inhibitory activity against rPfSUB-1 (data not shown).
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To evaluate any effect of the N-terminal His6 and S
tag-containing sequence on the inhibition mediated by rp31, we next
investigated the inhibitory properties of rp31SH, which comprises
exclusively the PfSUB-1 propeptide sequence
(Lys26Asp219). The determined
Ki(app) for inhibition by
rp31
SH was 12.5 nM (Table
I). This value is slightly higher than that found for rp31, but is
still in the low nanomolar range and confirms that the inhibition mediated by
rp31 is due to the propeptide component of its sequence. The difference
between this value and that found for intact rp31 is most likely the result of
experimental error in determination of the protein concentration of
rp31
SH; as shown in Fig.
1C (inset), there was a degree of unavoidable
heterogeneity in the rp31
SH preparation, presumably caused by limited
cleavage at secondary sites during digestion of rp31 with Factor Xa. The
presence of minor truncated species in the preparation that do not interact
with rPfSUB-1 would have the effect of lowering the specific activity of the
preparation. Additionally, any weakly interacting forms might compete with
intact rp31
SH for binding to the protease, also effectively leading to
an underestimate of the inhibition constant. Whatever the case, our data show
unambiguously that the PfSUB-1 propeptide is a fast binding, high affinity
inhibitor of rPfSUB-1.
We have previously shown that the Triton X-100-insoluble fraction of P. falciparum schizont extracts contains endogenous PfSUB-1 activity (11, 12). The addition of the unlabeled peptide substrate PEP1 to these extracts results in its conversion to two dominant cleavage products diagnostic of cleavage by PfSUB-1. Treatment of the schizont extract with purified rp31 was found to completely deplete the activity (Fig. 2C). This result shows that the activity of authentic, parasite-derived PfSUB-1 can be potently inhibited by the propeptide. This result is of particular interest because, whereas baculovirus-derived rPfSUB-1 is secreted in the p54 form, endogenous PfSUB-1 in schizont extracts is predominantly in the form of the terminal p47 processing product (11, 12). Our results therefore indicate that rp31 is able to bind to and inhibit both p54 and p47 forms of PfSUB-1, showing, in turn, that conversion of p54 to p47 does not per se abolish the ability of the protease to interact with its propeptide.
Selectivity of Inhibition by rp31The PfSUB-1 catalytic
domain is phylogenetically most closely related to the families of bacterial
subtilisins and bacterial pyrolysin subtilases
(8,
12,
27). Members of these families
sharing the highest amino acid homology with PfSUB-1 include subtilisin
BPN' and subtilisin Carlsberg (32 and 31% identities, respectively,
within the catalytic domains)
(12). To determine whether the
inhibitory activity of rp31 is selective for its cognate enzyme, we
investigated its effect on the activity of these two bacterial subtilisins in
kinetic assays by monitoring the effects of rp31 on hydrolysis of
Suc-AAPF-pNA as described under "Experimental
Procedures." Inhibition of both enzymes by rp31 was substantially poorer
than that seen with rPfSUB-1, with calculated
Ki(app) values of 0.33 ± 0.02
µM for subtilisin BPN' and 0.36 ± 0.05
µM for subtilisin Carlsberg. The experimentally determined
Km values for cleavage of Suc-AAPF-pNA
by these proteases under the conditions used were 242 ± 8
µM for subtilisin BPN' and 434 ± 51
µM for subtilisin Carlsberg (data not shown), so the above
Ki(app) values convert to
Ki values of 0.26 µM for
subtilisin BPN' and 0.32 µM for subtilisin Carlsberg
(22). Relative to inhibition
of rPfSUB-1 (Ki
5.3 nM), these
values correspond to selectivity indices of
49 and 60, respectively. Our
data suggest that rp31 is a relatively selective inhibitor of its cognate
enzyme.
Effect of Point Mutations and C-terminal Truncation on the Inhibitory Properties of rp31The S1 and S4 binding pockets are thought to dominate substrate preference in subtilases (27). Examination of the amino acid sequences flanking the cleavage sites used during autocatalytic processing of PfSUB-1 has revealed two conserved residues at the P1 (Asp) and P4 (Val) positions relative to the scissile bonds (11). Subsequent studies have shown that, although the P1 Asp is not crucial for substrate cleavage, it is the preferred residue at this position for efficient substrate cleavage in trans by PfSUB-1, and its replacement with Leu completely prevents cleavage (12). Similarly, the P4 Val plays an important role in substrate recognition (12). On the basis that these residues might also be important for the inhibition mediated by rp31, we generated, by mutagenesis, point mutations in appropriate positions of the recombinant propeptide. Three mutant forms of rp31 were investigated: a mutant with replacement of P4 Val216 with Ala (rp31-ASAD), a mutant with replacement of P1 Asp219 with Leu (rp31-VSAL), and a double mutant with replacement of both P1 Asp219 with Leu and P4 Val216 with Ala (rp31-ASAL) (Fig. 1; see Fig. 4A, third through fifth lanes).
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Inhibition assays with the mutant proteins showed that all remained potent
inhibitors of rPfSUB-1, with calculated
Ki(app) values of 5.1 ± 0.3,
2.6 ± 1.3, and 11.9 ± 3.6 nM for rp31-ASAD,
rp31-VSAL, and rp31-ASAL, respectively
(Table I). These results were
unexpected, indicating that the modified residues play little if any role in
the potent inhibition mediated by the propeptide. To further explore the
mechanism of inhibition by rp31, we therefore constructed, expressed, and
purified to homogeneity another construct identical to rp31 except for the
truncation of 11 residues (Leu209Asp219) from its
C terminus (Fig. 1; see
Fig. 4A, second
lane). Surprisingly, this construct (called rp3111) also inhibited
the activity of rPfSUB-1, although with a considerably reduced efficiency
compared with rp31 or the point mutants described above, with a calculated
Ki(app) of 76.6 ± 15.9
nM (Table I). This
indicates a requirement for interactions between the C-terminal
"tail" of the propeptide and PfSUB-1 for optimal inhibition, but
also shows that other interactions involving residues upstream of this domain
play an important role in binding of the propeptide to the catalytic
domain.
Stability of rp31 and rp31 MutantsThe propeptides of certain subtilases are readily and rapidly digested by their cognate enzymes. One practical consequence of this it that it can lead to potential inherent errors when attempting to experimentally define inhibition constants due to a time-dependent decrease in the concentration of the propeptide (e.g. Ref. 28). Although rp31 and its various mutants did not exhibit any discernible time-dependent decrease in their inhibitory capacity during the inhibition assays described above, we felt it important to assess the capacity of the propeptide to act as a substrate for rPfSUB-1 and to verify that the concentration of each polypeptide was stable during the assay. To assess this, purified rp31 or the various mutant propeptides were incubated at 37 °C in the presence of the substrate pepF1-6R and rPfSUB-1 under conditions identical to those used in the assays performed to measure their inhibitory activity. Two concentrations of each propeptide were tested, corresponding to the lowest and highest concentrations used in the inhibition assays. Fig. 3 shows that, even at the lowest propeptide concentration used, the concentration of each propeptide remained stable over time, and no digestion products could be detected upon SDS-PAGE analysis. Similar results were obtained using the highest concentration of each propeptide used in the inhibition assays (data not shown). These data clearly demonstrate that rp31 is completely resistant to digestion by PfSUB-1 over the time course of the assay. Moreover, the truncation and the various mutations made to the propeptide did not reduce this resistance to digestion.
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Binding of rp31 and rp31 Mutants to Baculovirus-derived PfSUB-1The above inhibition data clearly imply tight binding of rp31 and its various derivatives to rPfSUB-1. To directly visualize this, we used a modification of a binding assay described by Kessler and Safrin (29) and Markaryan et al. (30). The various recombinant propeptides were subjected to SDS-PAGE and electroblotted onto a membrane. The membrane was subsequently incubated with PBS containing purified rPfSUB-1 and then probed with a rabbit antiserum specific for the PfSUB-1 catalytic domain. A single band was observed in each case (Fig. 4B), and comparison with Coomassie Blue-stained duplicate gels showed that, in each case, the position of the band corresponded exactly to the position of migration of the corresponding propeptide construct (Fig. 4A). No signal was observed on control blots, which were not probed with rPfSUB-1 (Fig. 4C), demonstrating that the signals observed in Fig. 4B were not due to nonspecific recognition of the propeptides by the rabbit antibodies or the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies used. The specificity of PfSUB-1 binding was demonstrated using a set of standard molecular mass marker proteins, all loaded at a higher concentration than the propeptide constructs. Two species in the marker lanes, corresponding to phosphorylase b (94 kDa) and carbonic anhydrase (30 kDa), were visualized on the blot probed with rPfSUB-1 (Fig. 4B); however, both were also detected on the control blot (Fig. 4C), indicating that the recognition of these proteins was solely due to nonspecific binding of either the primary antibody or the horseradish peroxidase-conjugated secondary antibody and not to nonspecific binding of rPfSUB-1. Our results provide direct support for the above inhibition data, demonstrating that rPfSUB-1 is able to interact with all of the recombinant propeptide constructs in a highly specific manner.
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DISCUSSION |
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CD analysis of the recombinant propeptide both with (rp31) and without
(rp31SH) an N-terminal fusion peptide derived from the expression
plasmid demonstrated that it possesses secondary structure in the absence of
its cognate catalytic domain. Previous studies by others have indicated that
subtilase propeptides broadly divide into two groups according to their
folding capacity. Propeptides of bacterial subtilisin BPN' and
subtilisin Carlsberg are unfolded when expressed on their own
(17,
33,
34), whereas propeptides of
aqualysin I and of some of the subtilisin-like proprotein convertases (SPCs)
adopt significant secondary structure
(18,
35,
36). Thus, in the case of
aqualysin I, the isolated propeptide was estimated to be composed of 21%
-helix and 24%
-sheet
(35). The propeptides of
several SPCs have been shown to possess a similar degree of secondary
structure, with, for example, the propeptide of mouse SPC3 being 22%
-helical and 30%
-sheet
(18). The observed
-
content of rp31 is in good agreement with these values. It has
been suggested that propeptides with significant secondary structure are more
stable and more efficient in both folding and inhibiting the catalytic domain
than those that lack structure. In the case of subtilisin BPN', Kojima
et al. (33)
demonstrated, through a series of amino acid replacements, that an increase in
secondary structure in the propeptide could be correlated with a slower rate
of decrease in the inhibitory activity and a greater resistance to proteolytic
digestion in the presence of the cognate catalytic domain, both leading to a
lower Ki than that for the wild-type propeptide.
Likewise, the aqualysin I propeptide is more efficient in the folding and
inhibition of subtilisin BPN' compared with the subtilisin BPN'
propeptide itself, which lacks structure
(35). Unstructured propeptides
are thought to adopt structure only upon interaction with their cognate
catalytic domains (34,
37), so these observations are
consistent with a model in which possession of some pre-existing structure
accelerates productive binding to the catalytic domain because the structural
transition required for binding is minimized
(35). Our CD data indicate
that the PfSUB-1 propeptide belongs to the subclass of subtilase propeptides
that can fold in isolation from their cognate proteases, and our finding that
rp31 is a fast binding, high affinity inhibitor of rPfSUB-1 is consistent with
this.
Subtilase propeptides are not necessarily selective inhibitors of their cognate enzymes and, in some cases, are able to potently inhibit other subtilisins (14, 17, 18, 35). Indeed, the aqualysin I propeptide is a better inhibitor of subtilisin BPN' compared with the subtilisin BPN' propeptide (35), and the subtilisin E propeptide inhibits both subtilisin BPN' and subtilisin Carlsberg with Ki values in the low nanomolar range (18). The degree of sequence homology between bacterial propeptides correlates with inhibitory activity against the heterologous protease domain (e.g. Ref. 17). We observed 4960-fold higher Ki(app) values for inhibition of subtilisin BPN' and subtilisin Carlsberg by rp31 compared with its inhibition of PfSUB-1, suggesting that the propeptide has some selectivity for its cognate enzyme. There is no obvious sequence similarity between p31 and the subtilisin BPN' and subtilisin Carlsberg propeptides (8), so this result was not unexpected.
X-ray crystal structures of two bacterial subtilisin-propeptide complexes,
stabilized by appropriate mutation of the active-site serine, have shown that
the bound propeptide forms a structure consisting of two -helices and a
four-stranded
-sheet. This
-sheet packs against two parallel
surface
-helices of the catalytic domain, whereas the C-terminal tail
of the propeptide lies in a substrate-like manner within the activesite cleft
of the enzyme, blocking access of exogenous substrate. The complex is
stabilized by numerous hydrogen bonds between the two polypeptides, several of
which involve the four C-terminal residues of the propeptide tail in the
active-site cleft (38,
39). A recent NMR structure of
the 83-residue-long PC1 propeptide showed that the isolated molecule adopts a
fold similar to that of the enzyme-bound bacterial propeptide, despite the
absence of any sequence homology to the bacterial protein
(36). Homology modeling of the
PfSUB-1 catalytic domain has revealed an overall structure similar to that of
subtilisin BPN', including the presence of the two surface
-helices (12), so the
manner in which PfSUB-1 interacts with its propeptide is probably broadly
similar to that of the bacterial subtilisins. Our finding that point mutations
within the four residues at the C terminus of rp31 had little effect on its
inhibitory capacity and that even removal of 11 residues from the C terminus
did not abolish inhibition (although there was an
14-fold increase in
Ki(app)) was therefore unexpected. Our
data clearly demonstrate that, in the case of the PfSUB-1 propeptide,
interactions within the active-site cleft may be important for optimal
inhibition, but are not essential for complex formation. Since inhibition
presumably involves steric blockade of the PfSUB-1 active-site groove, it is
not clear how this is mediated by the truncated propeptide; however, it is
important to note that p31 is substantially larger than any of the subtilase
propeptides whose three-dimensional structures have been solved, and it is
possible that, in the complex formed between rPfSUB-1 and rp31
11,
structures formed by propeptide residues distant in the primary sequence from
its C terminus can occlude access to the active site.
Some subtilisin propeptides are efficiently degraded by their cognate
enzymes (e.g. Refs.
17 and
27). This can cause practical
difficulties in kinetic measurements, during which the propeptide
concentration needs to remain stable. It may also be important for eventual
autocatalytic removal of the propeptide from the protease catalytic domain
during activation. Our finding that rp31 was not degraded over the course of
the inhibition assays validates the accuracy of the
Ki(app) values calculated for all of
the propeptide constructs. It is also consistent with previous indications
that PfSUB-1 has a fairly stringent substrate specificity. Studies using
synthetic peptide substrates have indicated that the favored minimal
recognition sequence for PfSUB-1 is Val-X-X-Asp, where
X is any amino acid residue
(12). No such motif occurs
within p31 (besides that at the extreme C terminus). This apparent specificity
of PfSUB-1 raises the question of how activation of the protease ultimately
takes place during trafficking through the parasite secretory transport system
since it presumably requires removal of the propeptide. In many cases,
including that of the SPC furin, subtilase propeptide removal involves its
proteolytic cleavage, mediated by the cognate protease in cis or in
trans (40,
41). Our data suggest that
this is unlikely to be so in the case of Pf-SUB-1. We have previously shown
that, in the parasite, p54 is bound to its propeptide
(11) and that conversion of
p54 to p47 occurs only in a post-endoplasmic reticulum compartment
(8). Our demonstration here
that the propeptide can bind to and inhibit parasite-derived p47 implies that,
whatever the function of the conversion of p54 to p47, this processing step is
insufficient per se to remove the bound propeptide. Propeptide
release may be mediated by a simple environmental trigger (such as a change in
pH or calcium concentration) associated with transport beyond the parasite
endoplasmic reticulum.
The identity of the physiological substrate(s) of PfSUB-1 is unknown. Given the apparent high substrate specificity of the enzyme, it is unlikely to be involved in degradative processes; a role in specific post-translational protein modifications is more likely, which could take place either within the secretory organelles in which p47 accumulates or following protease release during erythrocyte invasion. We currently favor the former possibility since we have found that the addition of high concentrations of rp31 to P. falciparum cultures has no effect on the efficiency of erythrocyte invasion, and extensive experiments have indicated that rPfSUB-1 has no detectable capacity to modify surface proteins of the intact merozoite or the host red blood cell under nondenaturing conditions.2 In light of the findings described here, rp31 could be used to regulate the activity of PfSUB-1 in a specific and informative manner; it may be possible, for example, to subtly and specifically down-regulate levels of functional PfSUB-1 in the parasite by constitutive overexpression of the propeptide from a suitable transgene (42). The demonstration that rp31 inhibits both authentic and baculovirus-derived PfSUB-1 validates the use of the recombinant protease as a tool for high throughput screening for low molecular mass inhibitors (21). Defining the important inhibitory sequences in rp31 could aid in developing potent inhibitors with potential as antimalarial compounds. Indeed, endogenous inhibitors are generally a good starting point for further development of smaller but highly selective inhibitors (4347).
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 44-208-816-2127; Fax: 44-208-816-2730; E-mail: mblackm{at}nimr.mrc.ac.uk.
1 The abbreviations used are: PfSUB-1, P. falciparum subtilisin-like
protease-1; rPfSUB-1, recombinant PfSUB-1; rp31, recombinant p31; RP-HPLC,
reversed-phase high pressure liquid chromatography; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
Suc-AAPF-pNA,
N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide;
PBS, phosphate-buffered saline; SPC, subtilisin-like proprotein
convertase.
2 L. Jean and M. J. Blackman, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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