From the Protein Engineering Network of Centres of
Excellence and Department of Biochemistry, McGill University, Montreal,
Quebec H3G 1Y6, and the ¶ Pharmaceutical Biotechnology Sector,
Biotechnology Research Institute, National Research Council Canada,
Montreal, Quebec H4P 2R2, Canada
Received for publication, July 5, 2000, and in revised form, December 4, 2000
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The steps involved in the maturation of
proenzymes belonging to the papain family of cysteine proteases have
been difficult to characterize. Intermolecular processing at or near
the pro/mature junction, due either to the catalytic activity of active
enzyme or to exogeneous proteases, has been well documented for this family of proenzymes. In addition, kinetic studies are suggestive of a
slow unimolecular mechanism of autoactivation which is independent of
proenzyme concentration. However, inspection of the recently determined
x-ray crystal structures does not support this evidence. This is due
primarily to the extensive distances between the catalytic thiolate-imidazolium ion pair and the putative site of proteolysis near
the pro/mature junction required to form mature protein. Furthermore,
the prosegments for this family of precursors have been shown to bind
through the substrate binding clefts in a direction opposite to that
expected for natural substrates. We report, using cystatin C- and
N-terminal sequencing, the identification of autoproteolytic intermediates of processing in vitro for purified
recombinant procathepsin B and procathepsin S. Inspection of the x-ray
crystal structures reported to date indicates that these reactions
occur within a segment of the proregion which binds through the
substrate binding clefts of the enzymes, thus suggesting that these
reactions are occurring as unimolecular processes.
Prior to being shuttled to the mature lysosome, cysteine proteases
of the papain family are first synthesized as latent precursors of
higher molecular weight. Zymogens of papain-like enzymes are composed
of polypeptide extensions of various lengths at the N terminus of the
mature enzyme domain which act as potent inhibitors toward the cognate
enzyme (1-3). Because most precursors of the papain superfamily are
susceptible to autoactivation upon their exposure to acidic pH
environments (4-9), the stability of the prosegment1-enzyme complex is
believed to rely mainly on electrostatic interactions. The crystal
structures of rat (10) and human (11, 12) procathepsin B, human
procathepsin L (13), procaricain (14), human procathepsin K (15, 16),
and procathepsin X (17) have been reported recently. With the exception
of procathepsin X (17), each of these structures reveals that the
enzyme is inhibited by a small segment of the proregion which binds
through the substrate binding cleft in a direction opposite to that
expected for natural substrates. To protect cells from unregulated
digestion, this reverse configuration is believed to help ensure
proenzyme stability at neutral pH as the zymogen is passed from the
endoplasmic reticulum to its final destination, i.e. the
acidified lysosomal compartment of the cell.
In general, zymogens of all families of proteolytic enzymes may undergo
maturation using either intermolecular or intramolecular processing
pathways. For instance, autoproteolytic cleavage of prosubtilisin E, a
serine protease precursor, has been suggested to occur at the same site
near the pro/mature junction in either an intermolecular or
intramolecular manner, and the mechanism that predominates is dependent
mainly on the starting concentration of the proenzyme (18, 19). The
conformational rearrangements involved in the unimolecular mechanism of
prosubtilisin E, however, have yet to be elucidated. Similarly, kinetic
studies that monitor the conversion of propapain (4, 7), procathepsin B
(5), and procathepsin L (6) to active enzyme have revealed both an
intermolecular and intramolecular component to processing. Significantly, the kinetic studies (5-7) have also revealed that the
molecularity, i.e. the concentration of proenzyme at which the rate of the intermolecular events equals that of the unimolecular processes, is in the range of only 10 Interestingly, the proposal of a unimolecular step of maturation is
inconsistent to what is observed in the three-dimensional structures
for this family of precursors (10-16). For example, the crystal
structure of procathepsin B reveals that the pro/mature junction,
i.e. the final site for proteolytic processing, is ~28 Å
from the catalytic nucleophile. In addition, direct comparison of the
crystal structures of procathepsin B (10-12) with that of mature
cathepsin B (20) reveals no evidence for major N-terminal rearrangement
within the mature segment following the maturation process as is found
in zymogens belonging to other families (21-23). Hence, the crystal
structures reported to date have not independently provided any clues
to a plausible unimolecular step. Recently, a nonhomology
knowledge-based strategy predicted that a unimolecular proteolytic step
in propapain processing may involve the adjustment of a single Here, we attempt to identify novel intermediates of processing for the
precursors of cathepsin B and cathepsin S in vitro. This has
been achieved by monitoring using
SDS-PAGE3 the processing of
either purified procathepsin B or procathepsin S in the presence of the
endogenous inhibitor, cystatin C. Cystatin C has been shown to be a
substrate binding cleft-directed protein inhibitor of papain-like
enzymes with Ki values in the subnanomolar range
(27-29). Because the formation of a tight binding complex with
cystatin C requires that the substrate binding cleft of papain-like
enzymes be unobstructed (29), for example free from the prodomain, it
may be reasonably assumed that the affinity of cystatin C for the
mature enzyme would be superior to that for either the full-length
proenzyme or any intermediates generated during autoproteolysis,
i.e. hierarchy of cystatin C affinity for mature enzyme > intermediates > proenzyme. In its excess, therefore, cystatin
C may provide the desired effect of decreasing the rate of
intermolecular proteolytic processing, which is mainly the result of
the activity of mature (processed) enzyme, and thereby favoring the
detection of other processing events. Furthermore, we investigate a
role for the N terminus of the mature enzyme domain in the processing
of precursors belonging to the papain family. This has been achieved by
performing site-directed mutagenesis of Arg-8 to an alanine residue in
propapain, i.e. causing the destruction of the conserved
Asp-6 Materials--
Human wild-type cystatin C were a generous gift
from Dr. Irena Ekiel (Biotechnology Research Institute
(BRI)/National Research Council) and monoclonal antibody to
papain was provided by Daniel Tessier and Dr. David Y. Thomas
(BRI/National Research Council). Recombinant human wild-type
procathepsin B was expressed and purified as described previously (30).
The pPIC9 vector and Pichia pastoris strain GS115 were
purchased from Invitrogen (San Diego). Butyl-Sepharose resin was
purchased from Amersham Pharmacia Bio tech. The substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine
4- methylcoumarinyl-7-amide hydrochloride (Z-Phe-Arg-MCA) and the
irreversible inhibitor E-64 (L-trans-epoxysuccinyl)-L-leucyl-amino(4-guanidino)butane)
were purchased from IAF Biochem International Inc. (Laval, Canada). Polyvinylidene difluoride (PVDF) membranes were purchased from Applied Biosystems.
Expression of Procathepsin B and Procathepsin S--
A cDNA
construct consisting of human wild-type procathepsin B or procathepsin
S as a fusion with the preproregion of yeast Purification of Procathepsin B and Procathepsin S--
The
proenzymes were purified from the culture supernatant using a
hydrophobic resin under nonacidic conditions. The culture supernatant
(250 ml) was concentrated to 50 ml using an Amicon stirred cell (YM-10
membrane). During concentration, the supernatant was exchanged to 50 mM Tris (pH 7.4) containing 1.6 M
(NH4)2SO4. Concentrated recombinant
proenzyme was then purified on a fast protein liquid chromatography
system (Amersham Pharmacia Biotech) using a butyl-Sepharose fast flow
column. Proenzyme fractions eluted from the column by applying a linear
gradient of decreasing ammonium sulfate concentration. Glycosylated
procathepsin B and procathepsin S eluted at 0.6-0.8 M and
0.3-0.5 M (NH4)2SO4,
respectively, and samples were stored at 4 °C.
In Vitro Processing of Procathepsin B and Procathepsin
S--
Purified procathepsin B (20 µM) and procathepsin
S (4 µM) samples were dialyzed against 50 mM
sodium acetate (pH 5.0), 1 mM dithiothreitol at 4 °C
overnight in the presence (or absence) of 100 µM human
wild-type cystatin C. Each sample was then treated with excess E-64
followed by the addition of reducing buffer and denaturation in a
boiling water bath. Protein samples were then applied to SDS-PAGE (12%
gels) and stained with Coomassie Brilliant Blue R-250 (Bio-Rad) or
AgNO3 (see Fig. 1).
Expression and Processing of Wild-type and R8A
Propapain--
Propapain was produced as described previously (4, 7).
Briefly, the Saccharomyces cerevisiae strain BJ3501 was
transformed with the expression vector derived from pVT100-U in which
the propapain gene is under the control of the N-Terminal Identification of Protein Bands--
Following
SDS-PAGE, protein bands were blotted onto hydrophobic PVDF membranes.
The membranes were then stained with Coomassie Brilliant Blue
R-250, and each protein band of interest was subjected to a
minimum of five cycles of automated Edman degradation using the method
described previously (31).
Fluorogenic Assay for Monitoring Proenzyme
Processing--
Processing of human wild-type procathepsin B and
procathepsin S was followed in a continuous manner by carrying out the
reactions in a 3-ml quartz cuvette in the presence of the substrate
Z-Phe-Arg-MCA (10 µM) and measuring fluorescence as a
function of time. The conversion of procathepsins B and S to active
enzyme leads to hydrolysis of the substrate, and fluorescence of the
MCA product was monitored using excitation and emission wavelengths of
380 and 440 nm, respectively. Processing was initiated by lowering the
pH from 7.4 (pH of the stock solution of procathepsins B and S) to 5.0 (pH of the assay). Reactions were carried out at 25 °C in the
presence of 50 mM sodium acetate buffer, 0.2 M
NaCl, 2 mM EDTA, 2 mM dithiothreitol, and 3%
dimethyl sulfoxide. The reaction mixture was stirred continuously in
the cuvette during the reaction. The product versus time
curves were fitted to the following equation (32-36).
Identification of Intermediate Cleavage Sites in Procathepsin B
Using the Cystatin C Assay--
On addition of cystatin C to a
reaction mixture with which the processing of procathepsin B is being
monitored, an intermediate species of cathepsin B is observed. This
intermediate is observable even at nanomolar concentrations of
proenzyme (as determined by AgNO3-stained SDS-PAGE; Fig.
1) as well as at micromolar concentrations (Fig. 2A; PVDF membranes,
Coomassie Blue staining). The ability to immobilize on PVDF membranes,
and consequently purify, protein bands that correspond to presumably
unstable processing intermediates ensures that the residual prosegment
of these species remains resistant to further degradation. Direct
N-terminal sequencing of the intermediate band of cathepsin B
processing (Fig. 2A) indicated a mixture of intermediate
species with autocatalytic cleavage having taken place at
Cys-42p Identification of Intermediate Cleavage Sites in Procathepsin S
Using the Cystatin C Assay--
In the case of procathepsin S, the
presence of cystatin C is not strictly required to observe, using
SDS-PAGE, the accumulation of an intermediate processing band that is
~2 kDa heavier than the mature enzyme (data not shown). To ensure the
accumulation of sufficient quantities of this species for N-terminal
identification, cystatin C was added to procathepsin S (Fig.
2B) under processing conditions to inhibit bimolecular
reactions as has been discussed previously for procathepsin B. Based on
their migration on SDS-PAGE, it is assumed that the intermediate
species formed in the presence of cystatin C are identical to those in
the absence of the inhibitor. This protein band obtained for the sample
treated with cystatin C was found to be a mixture of species
corresponding to cleavage at Ser-76p Continuous Monitoring of Wild-type Procathepsin B and Procathepsin
S Autocatalytic Processing at pH 5.0--
A continuous assay based on
the hydrolysis of the substrate Z-Phe-Arg-MCA by the active enzyme
generated in the autocatalytic process was used. The rate of substrate
hydrolysis increases with time due to time-dependent
release of active enzyme from the precursor until a constant rate is
obtained which corresponds to the activity of fully processed enzyme.
The curves can be fitted to a model that assumes a first-order increase
in rate from an initial rate vPE, corresponding
to the activity of the precursors (if any), to a final rate
vE, corresponding to the activity of fully
processed enzyme. Based on the results of nonlinear regression
analysis, no significant activity of the precursors against the
Z-Phe-Arg-MCA substrate could be detected, and the first-order rate for
autocatalytic processing, kobs, increases
linearly with proenzyme concentration (Fig.
4). The direct link between the rates of
autoprocessing and precursor concentration confirms the occurrence of a
bimolecular reaction, i.e. intermolecular processing of
proenzyme by fully or partially processed (active) enzyme. In support
of the postulated unimolecular autoproteolytic reactions discovered
using the cystatin C assay (discussed above), the extrapolated rate
constants were calculated to be 2.5 × 10 Role of the N Terminus of the Mature Segment in the Autocatalytic
Processing of Precursors Belonging to the Papain
Family--
Site-directed mutagenesis was performed to test the
hypothesis that a rearrangement of the N terminus of the mature domain of cysteine proteases is involved in an intramolecular processing mechanism. Specifically, to remove the salt bridge that is formed between the highly conserved residues Asp-6 and Arg-8 an R8A mutant of
propapain was produced. From the data in Fig.
5 we conclude that R8A propapain remains
competent to autoactivate to form mature protein.
The crystal structure of procathepsin B (10-12) indicates that
the Leu-41p
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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9
M. Thus it is only at very low concentrations of the
proenzymes that the unimolecular mechanism plays a significant role in processing.
-turn
which rearranges the first 12 residues2 of the enzyme domain
and allows the mature N terminus to reach the active site in a
cleavable direction (24). Within this segment of the mature N terminus
are found Asp-6 and Arg-8, which are both highly conserved
residues among cysteine proteases of the papain family (25).
Interestingly, all x-ray crystal structures of papain-like enzymes
(pro- and mature) reported to date reveal that the side chains of Asp-6
and Arg-8 contribute to the formation of a conserved salt bridge
(10-17, 20, 26) that contributes to the structural integrity of the
mature enzyme's N terminus.
Arg-8 salt bridge, and monitoring the overall effect of this
mutation on the ability of propapain to
autoactivate.4
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-factor was digested
with XhoI and NotI, and the proenzyme fragment was subcloned into the pPIC9 vector (Invitrogen). For integration into
the Pichia genome, the pPIC9-based constructs were
linearized by cleavage with BglII and purified. The P. pastoris host strain GS115 (Invitrogen) was then transformed with
the linearized constructs by electroporation. Positive transformants
were grown for 2 days at 30 °C in medium containing glycerol as the
carbon source followed by incubation in the presence of methanol for a
further 3 days to induce expression of recombinant protein. The
consensus sequence for oligosaccharide substitution located on the
occluding loop within the mature enzyme domain of cathepsin B had been
removed by the substitution S115A. All other sites for
oligosaccharide substitution within procathepsin B and procathepsin S
were left unaltered. Protein secreted into the culture supernatants was analyzed by SDS-polyacrylamide gel electrophoresis (12% gels).
-factor promoter. Yeast cells were first grown under selective conditions to ensure plasmid maintenance and then transferred into a rich medium. The cells
were lysed using a French pressure cell, and the soluble fraction of
the crude lysate included propapain. Propapain was then partially
purified using butyl-Sepharose resin (Amersham Pharmacia Biotech) as
discussed previously for procathepsins B and S. Complete processing
in cis was achieved by incubating propapain at 65 °C in
50 mM sodium acetate (pH 3.8), 20 mM cysteine
for 30 min. Samples were then analyzed by Western blot following
separation of the proteins using SDS-PAGE.
where P is the MCA product formed, vPE
represents the initial rate of product release (which should reflect
activity of the proenzyme, if any), vE
corresponds to the rate for mature (active) enzyme, and
kobs is a first-order rate constant.
(Eq. 1)
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Gly-43p (70% of total signal) and Arg-40p
Leu-41p (30% of
total signal) (Fig. 3, cathepsin B prosegment numbering). After several weeks of incubation in the presence of
cystatin C, both full-length procathepsin B and the processing intermediates disappear, and only the protein band corresponding to
mature cathepsin B is observed. Therefore, it may be concluded that
these are true intermediates of processing and not dead-end (side
product) reactions. Because complete maturation of cathepsin B has been
shown to also include autocatalytic trimming of six residues at the
C-terminal end of the enzyme (37), it should be noted that the 30- and
32-kDa protein bands in Fig. 2A, lane 2, most
likely correspond to cathepsin B composed of a mature and semimature N
terminus, respectively, and an unprocessed C terminus.
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Fig. 1.
SDS-PAGE stained with AgNO3.
Procathepsin B (37 kDa) at low concentrations (2 nM) was
exposed to 50 mM acetate buffer (pH 5.0) and 1 mM dithiothreitol for 12 h in the presence of 10 nM (lane 1), 50 nM (lane
2), and 0 nM (lane 3) human cystatin C. Note that the proportion of processing intermediate (32 kDa) to mature
cathepsin B (30 kDa) is improved with increasing concentration of
cystatin C.
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Fig. 2.
PVDF membranes (Applied Biosystems, Problott
TM membranes) stained with Coomassie Brilliant
Blue R-250 (Bio-Rad) containing immobilized procathepsin B (panel
A) and a mixture of glycosylated and deglycosylated
procathepsin S (panel B). Lanes 1 and
2 of each membrane represent the conversion of proenzyme to
mature enzyme in the absence and presence of 100 µM
cystatin C (Mr = 12.5 kDa), respectively. The
processing intermediates migrate at 32 and 29 kDa for procathepsin B
and procathepsin S, respectively. The 29-kDa species for procathepsin S
is detectable in low quantities using SDS-PAGE in the absence of
cystatin C (data not shown).
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Fig. 3.
Structure-based sequence homology of the
C-terminal ends of the prosegments of cathepsin B and cathepsin S. Cathepsin B prosegment numbering was used for cathepsin B, and
cathepsin S prosegment numbering was used for cathepsin S. The
established cleavage sites to form mature protein near the pro/mature
junction are represented as . The sites of autoproteolytic
processing detected using the cystatin C assay are denoted as
;1.
Ser-77p (50% of total signal)
and Met-72p
Ser-73p (50% of total signal) (Fig. 3; cathepsin S
prosegment numbering).
4 s
1 and 8.2 × 10
3 s
1 for
procathepsin B and procathepsin S, respectively, as the concentration of these precursors approach zero. This kinetic data support the involvement of an intramolecular event in the processing of these precursors which is independent of the concentration of proenzyme.
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Fig. 4.
Continuous assay for the autocatalytic
processing of wild-type procathepsin B (panel A) and
procathepsin S (panel B). Shown are plots of the
first-order rates of processing (kobs) obtained
by nonlinear regression of the data discussed in Equation 1 under
"Experimental Procedures" as a function of precursor concentration
(determined by active site titration with the E-64 inhibitor). The data
are in agreement with a first-order rate of processing. For both
procathepsins B and S, the rate of processing,
kobs, increases linearly with proenzyme
concentration, indicative of a bimolecular reaction that most likely
corresponds to intermolecular processing of proenzyme by mature or
semimature protein. In addition, there is a corresponding rate constant
of precursor activation as the concentration of proenzyme is
extrapolated to zero (2.5 × 10 4
s
1 for procathepsin B and 8.2 × 10
3 s
1 for
procathepsin S), indicative of an activation event that is independent
of the concentration of precursor.
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Fig. 5.
Monitoring the autocatalytic processing of
wild-type propapain (lane 1) and R8A propapain
(lane 3) using Western blot analysis following their
incubation at 65 °C in 50 mM
acetate buffer (pH 3.8) and 20 mM cysteine for 30 min
(lane 2, wild-type mature papain; lane
4, R8A mature papain). Molecular masses of the
papain precursor and mature form are 37 and 25 kDa, respectively. The
R8A variant of propapain was observed to autoprocess as efficiently as
the wild-type precursor.
DISCUSSION
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DISCUSSION
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Gly-47p segment of the cathepsin B proregion binds through the active site cleft of the enzyme in the reverse substrate binding mode and in an extended conformation. These structures also
reveal that the carbonyl carbon of Cys-42p is in closest proximity to
the catalytic residue (Fig. 6) (note that the
coordinates illustrated in Fig. 6 are those for C29S human procathepsin
B at pH 5.7 (11, 12) and not the wild-type precursor). Inspection of
this region of the cathepsin B precursor indicates that the carbonyl
carbon of Cys-42p is located ~4.3Å from the catalytic nucleophile
and the potential bond angle between the catalytic nucleophile and the
carbonyl oxygen of Cys42p is 131°, i.e. conducive to
forming a tetrahedral intermediate (Fig. 6). For the Arg-40p
Leu-41p cleavage site, the distance and potential bond angle between the carbonyl group of Arg-40p and the catalytic nucleophile are ~6.9 Å and 45°, respectively. Hence, the ability of the carbonyl carbon of
Arg-40p to reach the catalytic center of the enzyme for hydrolysis would suggest that significant conformational mobility exists within
the segment composed of residues Asp-34p
Leu-41p which interact
with the occluding loop crevice (10) as well as the primed subsites of
the substrate binding cleft of cathepsin B.
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Fig. 6.
Active site cleft in human
C29S procathepsin B at pH 5.7 (11, 12). The cathepsin B prosegment
(red residues) binds through the substrate binding cleft of
cathepsin B (blue residues) in the reverse N C direction
to that taken by natural substrates. The carbonyl carbon of Cys-42p is
in closest proximity to the catalytic nucleophile (4.3 Å), and the
potential bond angle between the catalytic nucleophile and the carbonyl
oxygen of Cys42p is 131°.
Given the proximity of these carbonyl carbons (those of Cys-42p and
Arg-40p) to the catalytic center, it is tempting to speculate that
these reactions occur in an intramolecular manner. As discussed previously, kinetic studies are suggestive of a unimolecular step among
members of the papain family of precursors whose molecularity is
unusually low (109 M) yet is
still much faster than noncatalyzed (spontaneous) peptide hydrolysis.
The low molecularity may be accounted for by the reverse binding mode
adopted by the prodomain in its interaction with the active site cleft
of the enzyme. As a consequence of the reverse substrate binding mode,
the formation of a tetrahedral intermediate at the carbonyl carbon of
Cys-42p or Arg-40p would not be stabilized by the oxyanion hole formed
by Gln-23. This structural incompatibility has, therefore, led to the
suggestion that it would not be possible for the enzyme to perform such
reactions (12). Previous work with oxyanion hole mutants of papain (38)
and cathepsin B,5 however,
indicates that the oxyanion hole is not an absolute requirement to
hydrolyze small synthetic substrates but rather is a feature that may
improve the catalytic efficiency of these enzymes by only 10-100-fold
depending on the substrate under study. Alternatively, it may be
proposed that the slow rate of these unimolecular reactions may be
because the reverse complementarity between the bound prosegment and
the enzyme's substrate binding cleft causes the distance between the
N of the catalytic His-199 and the backbone amide (leaving)
group of either Gly-43p or Leu-41p to be larger than would be the case
for natural substrates (39, 40). Hence, protonation from the
N of
the catalytic histidine to the prosegment bound in the reverse mode is
likely to be less efficient to that for substrates bound in the usual
substrate binding mode, thus resulting in a reversible nucleophilic
reaction that has difficulty going to completion. Because the catalytic thiol among cysteine proteases is more nucleophilic and constitutes a
better leaving group than the catalytic oxygen found among serine proteases, it has been postulated that proton transfers effectuated by
the catalytic histidines found among cysteine proteases would need to
be more efficient than those found among serine proteases (41),
i.e. the catalytic histidine found in cysteine proteases must compensate for the lower pKa of the catalytic
thiol group.
The ability to detect cleavage at the carbonyl carbon of Arg-40p
indicates the existence of a significant degree of conformational mobility for the proregion within the prosegment-substrate binding cleft interface. This mobility may be accounted for by the
pH-dependent stability of the enzyme's occluding loop,
which consequently defines the pH-dependence of propeptide binding as
well as the overall rate of procathepsin B processing (30). Competition
between the prosegment and the occluding loop for the surface of the
enzyme termed the occluding loop crevice (10) was shown to
be regulated by the formation of a critical salt bridge between His-110
of the occluding loop and Asp-22 located within the primed subsites of
the enzyme's active site cleft. Site-directed mutagenesis of either
one of His-110 or Asp-22 to an alanine residue produces a variant of
procathepsin B which is stable and incapable of autoactivation. Remaining elusive from these studies (30), however, was whether these
mutations caused the perturbation of a unimolecular event involving
proteolysis of the prosegment. As procathepsin B is exposed to acidic
pH conditions, it is possible that salt bridge formation between
His-110 and Asp-22 promotes competition between the occluding loop and
the N-terminal -helical cap of the proregion for the occluding loop
crevice. From this competition, it follows that the remaining
C-terminal residues of the proregion, i.e. prosegment
residues that stretch from the substrate binding cleft to the
pro/mature junction, would have reduced affinity for the surface of the
enzyme and increased conformational mobility. In agreement with this
proposal is the consistent lack of secondary or tertiary structure
found within the C-terminal end of papain-like prosegments when bound
to the cognate enzyme (10-17). Furthermore, truncated propeptides
composed only of these C-terminal residues display significantly weaker
affinity for the enzyme than the full-length propeptide (42-43).
Additional evidence for mobility within the prosegment has been shown
for the propeptide of cathepsin L which loses most of its tertiary
structure yet almost none of its secondary structure at low pH (44). It
is believed the high B-factors corresponding to the C-terminal end of
papain-like prosegments facilitate the autocatalytic conversion of
these zymogens upon their exposure to acidic pH. That the
conformational mobility within the C-terminal end of papain-like
prosegments is important for autoprocessing is evidenced by the results
that have been obtained recently for procathepsin
H.6 Because of the
predicted preformation of a disulfide bond linking the C-terminal end
of the cathepsin H prosegment to the main body of the enzyme, the
pro/mature junction within the cathepsin H precursor was found to be
highly resistant to proteolysis either in cis or
in trans.6
Based upon the sequence homology between procathepsin S and other cysteine protease proforms for which structural information is available (10-16), with the exception of procathepsin X (17), it has been possible to model the structure of the relevant portion of the procathepsin S proregion. Based on this model the predicted structure-based sequence homology between the prosegments of procathepsin B and procathepsin S is presented in Fig. 3, and it reveals that cleavage at the carbonyl carbon of residues in position 42p of procathepsin B and 76p of procathepsin S are well aligned. This conservation is observed despite the difference in length for these two prodomains, i.e. 62 residues for procathepsin B and >90 residues for procathepsin S, which is a member of the cathepsin L subfamily. It follows, therefore, that Ser-76p is predicted to bind through the substrate binding cleft of cathepsin S in the reverse binding mode and that its carbonyl carbon is located closest to the catalytic residue, Cys-25, as has been documented for Cys-42p in procathepsin B.
Cathepsin S prefers to cleave at internal sites within its prosegment
where serine residues are located in the S1' position, namely at the putative Lys-91pSer-92p site near the pro/mature junction needed to form mature cathepsin S as well as at the
Met-72p
Ser-73p and Ser-76p
Ser-77p sites identified using the
cystatin C assay (Fig. 3). It is interesting to note that two
consecutive serine residues are located within the prosequence of
cathepsin S, namely Ser-76p and Ser-77p. Curiously, proteolysis was
only observed at the carbonyl carbon of Ser-76p and not at the carbonyl
carbon of Met-75p. This result indicates that cleavage at the carbonyl carbon of Ser-76p may be selective. Similar to what was observed for
procathepsin B, cleavage at the carbonyl carbon of Met-72p suggests
that a significant amount of conformational mobility exists for
prosegment residues that bind through the substrate binding cleft of
cathepsin S. It is interesting to note that the conversion of
procathepsin H to its mature form has been proposed to involve cleavage
at the carbonyl carbon of Ser-77p (26) (cathepsin H prosegment
numbering), which is located adjacent to the cleavage sites identified
in this study for procathepsin B and procathepsin S using the cystatin
C assay. This cleavage site has been proposed to be a prelude to the
formation of an N-linked glycosylated octapeptide of
prosegment residues composed of Glu-78p
Thr-85p (cathepsin H
prosegment numbering), termed the mini-chain, which remains attached to mature cathepsin H via a disulfide bond (26).
Is it possible that the processes identified by the cystatin C assay are the result of intermolecular reactions caused by catalytic (undetectable) amounts of mature enzyme or activated proenzyme? If processing were assumed to be solely the result of intermolecular processes, then it would be expected that the conversion of full-length proenzyme to processing intermediates would be inhibited as efficiently by cystatin C as the conversion from processing intermediate to mature enzyme or the direct maturation of full-length proenzyme to mature protein. In the presence of cystatin C, however, this is not what is observed, but rather the time-dependent accumulation of intermediate protein bands is detected using SDS-PAGE.
Previous to this work, a nonhomology knowledge-based prediction of
propapain activation proposed that an intramolecular proteolytic event
may involve the N terminus of the mature enzyme domain moving toward
the active site cleft, thus facilitating the release of the prosegment
(24). Using the structure of mature papain as a template,
i.e. the effect of the prosegment was not considered, the
adjustment of a single -turn was postulated to permit the extension
of the first 12 residues at the N terminus of the enzyme and predicted
to allow the pro/mature junction to reach the active site in the
cleavable direction, i.e. the substrate binding mode. For
this rearrangement to be made possible, it would be expected that the
integrity of the salt bridge formed by Asp-6 and Arg-8 found in all
structures of papain-like enzymes reported to date (10-17, 20, 26,
45), i.e. residues that contribute to the
-turn, would
influence the overall pH-triggering mechanism of propapain processing.
However, as reported above, the removal of the salt bridge in the
papain mutant R8A does not influence the ability of propapain to
autoactivate to form mature protein (Fig. 5). These results collaborate
with the x-ray crystal structures of papain-like enzymes (pro- and
mature enzymes) which demonstrate high resolution among residue side
chains located at the N terminus of the mature segment, thus
corresponding to a region of the molecule which is conformationally
constrained (low B-factors). The N terminus of the mature segment
within the precursors (10-17) is in a conformation that is essentially
identical to that found in the crystal structures of mature enzyme (20,
26, 45), thus suggesting that no major N-terminal rearrangement is
observed during precursor activation. Furthermore, the overall
assumption that the putative site of proteolysis to form mature protein
near the pro/mature junction is the only possible cleavage site,
i.e. as has been proposed for prosubtilisin E (18, 19),
remains speculative as an unidentified processing intermediate was
observed for propapain at 30 kDa (7).
Nature of the Steps Involved in the Autocatalytic Processing of Procathepsin B and Procathepsin S-- Using site-directed mutagenesis, previous studies have established that the reactivity of the catalytic cysteine residue found within the precursors of papain-like enzymes is responsible for the maturation of this family of zymogens (4-9). Hence, autoactivation of zymogens belonging to the papain family requires that the precursors be composed of a preformed and functional catalytic center and substrate binding cleft. In this study, we have described the identification of novel processing intermediates for procathepsin B and procathepsin S. The intermediates identified for cathepsin B are observable only in the presence of cystatin C, whereas for cathepsin S, the intermediates are weakly observable on SDS-PAGE in the absence of cystatin C (data not shown), and their identification is facilitated only by the addition of cystatin C. That these novel cleavage products in procathepsin B and procathepsin S are observed at all starting concentrations of proenzyme, including very low concentrations, suggests that these reactions are occurring as unimolecular processes and that they may be important. The crystal structures of precursors of the papain family (10-16) demonstrate that these cleavage reactions are taking place within a segment of the proregion which binds through the active site cleft of the enzyme in the reverse substrate binding mode. Thus in effect the intramolecular processing of the precursors is analogous to the cleavage of a polypeptide chain (substrate) when bound in the reverse mode to the active site. To the authors' knowledge this is the first reported example of the observation of a protease cleaving a peptide bond in the reverse direction. It is interesting to note that although the rate of cleavage of the reversed amide bond is much slower than that of an optimally oriented bond, it is nonetheless several orders of magnitude faster than noncatalyzed hydrolysis.
Following the completion of the intramolecular proteolytic step, more than 20 residues derived from the C-terminal end of the prosegment continue to remain covalently attached to the mature segment via the pro/mature junction. Intuitively, these intermediate species would be as catalytically competent as the mature enzyme because propeptides composed of amino acid sequences corresponding to the C-terminal end of papain-like prosegments possess low inhibitory activity compared with that of the full-length propeptide (2, 42, 43).
Given the kinetic and structural data presented here, it is tempting to speculate that these reactions are occurring as slow unimolecular steps that may be necessary for triggering the intermolecular proteolytic cascade, i.e. the first step may involve the slow intramolecular cleavage reactions presented here, followed by the rapid intermolecular proteolytic cascade performed by the catalytic activity of mature or semimature species whose quantities accumulate with time. From this study, it may also be concluded that the N terminus of the mature domain does not participate through a conformational rearrangement in the pH-dependent autoprocessing mechanisms of zymogens belonging to the papain family.
It has been proposed that deregulated secretion of papain-like enzymes
to the extracellular matrix may serve as the catalyst for propagating
several disease states. For example, cathepsin B has been implicated in
tumor metastasis (46) as well as rheumatoid arthritis (47). In chronic
inflammatory disease, a degradative phenotype of monocyte-derived
macrophages have been shown to secrete enzymically active forms of
cathepsins B, L, and S into the extracellular milieu (48). Furthermore,
deregulated cathepsin K activity has been linked to osteoporosis (49).
Because the prodomains are known to act as intramolecular chaperones
(4), it may be reasonably assumed that these enzymes are targeted to
the extracellular matrix in their precursor form. Despite the neutral
pH environment generally associated to the extracellular matrix, it has
been postulated that microenvironments of acidic pH may allow low
concentrations of zymogens of papain-like enzymes to be converted to
their mature forms. The discovery of a unimolecular mechanism of
processing for this family of precursors may help to explain how
lysosomal enzymes (even at low concentrations) have been implicated in
a number of degradative and invasive pathological conditions
extracellularly. Although inhibitors with sufficient potency are
available for this class of enzymes, they often lack the required
selectivity needed for therapeutic applications. In addition to the
traditional approach of designing substrate binding cleft-directed
inhibitors, an improved understanding into the molecular basis of
autoprocessing for zymogens of the papain family may lead to novel
therapeutic strategies in which the conversion of proenzyme is
intervened. This would have significant consequences given that the
prosegments of papain-like cysteine proteases are intrinsic appendices
that are potent inhibitors of the enzyme from which they originate.
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ACKNOWLEDGEMENTS |
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We thank France Dumas for the N-terminal sequence determinations and Dr. Irena Ekiel for her generous gift of human wild-type cystatin C. We also thank Daniel Tessier and Dr. Dave Thomas for providing the constructs of wild-type and R8A propapain, and Dr. Jayaraman Sivaraman for help in preparing Fig. 6 depicting the active site cleft of procathepsin B.
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FOOTNOTES |
---|
* 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.This
work was funded in part by the Government of Canada Network of Centres of Excellence Program supported by the Medical Research Council of Canada and the Natural Sciences and Engineering Research Council of Canada through the Protein Engineering Network of Centres of Excellence, Inc. This is National Research Council of Canada Publication 42997.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.
§ Present address: Dept. of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, 16711 Trans-Canada Highway, Kirkland, Quebec H9H 3L1, Canada.
To whom correspondence should be addressed: Pharmaceutical
Biotechnology Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Tel.: 514-496-6256; Fax: 514-496-1629; E-mail: Andrew.Storer@nrc.ca.
Published, JBC Papers in Press, December 13, 2001, DOI 10.1074/jbc.M005851200
1 In the text, the words prosegment, proregion, prosequence, and prodomain refer to the polypeptide stretch located N-terminal to the mature enzyme in the proenzyme; the word propeptide refers to the chemically synthesized polypeptide corresponding to the proregion sequence but without the mature enzyme.
2 In the text, residue numbering relates to that of cathepsin B for recombinant human cathepsin B, and to that of cathepsin S for recombinant human cathepsin S. Residues in the proregion are identified with the suffix p.
4 In the text, the terms autoprocessing, autoactivation, and maturation relate to the ability of zymogen to convert to mature protein at acidic pH due to autocatalytic cleavage at or near the pro/mature junction.
5 D. K. Nägler, A. C. Storer, and R. Ménard, manuscript in preparation.
6 O. Quraishi, R. Ménard, J. S. Mort, and A. C. Storer, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; E-64, trans-epoxysuccinyl-L-leucyl-amido-(4-guanidino)butane; Z-Phe-Arg-MCA, benzyloxycarbonyl-L-phenylalanyl-L-arginine 4-methylcoumarinyl-7-amide; PVDF, polyvinylidene difluoride.
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