From The Burnham Institute, La Jolla, California
92037, the § Department of Protein Design, Novo Nordisk,
DK-2880 Bagsvaerd, Denmark, the ¶ Department of Biochemistry and
Biophysics, University of California, San Francisco, California 94043, and the
Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, Georgia 30602 and the Faculty of
Biotechnology, Department of Microbiology, Jagiellonian University,
Krakow 30-060, Poland
Received for publication, October 15, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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Most proteases are synthesized as inactive
precursors to protect the synthetic machinery of the cell and allow
timing of activation. The mechanisms used to render latency are varied
but tend to be conserved within protease families. Proteases belonging
to the caspase family have a unique mechanism mediated by transitions of two surface loops, and on the basis of conservation of mechanism one
would expect this to be preserved by caspase relatives. We have been
able to express the full-length precursor of the Arg-specific caspase
relative from the bacterium Porphyromonas gingivalis, Arg-gingipain-B, and we show that it contains N- and C-terminal extensions that render a low amount of latency, meaning that the zymogen is substantially active. Three sequential autolytic
processing steps at the N and C terminus are required for full
activity, and the N-propeptide may serve as an intramolecular chaperone rather than an inhibitory peptide. Each step in activation requires the
previous step, and an affinity probe reveals that incremental activity
enhancements are achieved in a stepwise manner.
Proteases of the gingipain family are virulence factors of the
periodontal pathogenic bacterium Porphyromonas gingivalis
(1-3). This group contains two genes that encode Arg-specific
proteases (rgpA and rgpB) and one gene encoding a
Lys-specific protease (kgp). Gene ablation studies have
shown that RgpA and RgpB are required for the activation of Kgp,
placing the Arg-specific proteases at the top of a proteolytic pathway
required for bacterial growth (4, 5). The protein encoded by
rgpB is predicted to consist of three distinct segments (see
Fig. 1), but only two are found in the mature product isolated from
bacterial cultures, the catalytic unit and an Ig domain. Consequently
the 205-amino acid N-terminal segment may constitute an activation
peptide that restrains the activity of the protease until it reaches
its site of action.
Structural analysis of the catalytic unit of RgpB (6) demonstrates that
it shares its evolutionary origin with a common ancestor of caspases,
proteases involved in apoptosis and cytokine activation (7). Moreover,
homology mapping suggests that the clan encompassing gingipains and
caspases also may contain bacterial clostripain, plant and animal
legumains (processing proteases) (8), and separase (required for sister
chromatid separation during anaphase) (9). This clan is known as
protease clan CD (10) or the caspase-hemoglobinase fold (11).
The majority of proteases are synthesized as zymogens that await
activation at a suitable time to protect the biosynthetic machinery of
the cell against activation and to act as a timing event in biological
function (12). Thus, one of the key events in any proteolytic pathway
is the conversion of the zymogen to the active enzyme. Different
protease clans utilize distinct strategies for zymogen maintenance and
activation, but within clans there seems to be conservation of a
particular strategy. On the basis of conservation of mechanism one
would imagine that protease clan CD would embrace a similar activation
pathway, meaning that the zymogens of caspases and RgpB should be
stabilized by homologous molecular interactions. The molecular
determinants of caspase activation have been elucidated (13-15), and
this seems not to involve the removal of N-terminal segments common for
other protease clans (12).
Consequently the understanding that the precursor of RgpB (pro-RgpB)
may require truncation at its N terminus (or even C terminus) for its
activation serves as a good model to test the conservation hypothesis
for protease zymogen activation since these observations would seem to
contrast with the caspase activation mechanism. This study presents a
detailed investigation of the autocatalytic processing of recombinant
pro-RgpB, including the characterization of intermediates on the
activation pathway, to clarify the mechanism of pro-RgpB maturation.
Strains and Media--
Saccharomyces cerevisiae
strain YG227
(Mata, Plasmid Construction--
The pRS316Gal( Production of Recombinant Progingipain in Yeast
Cells--
Yeast cells were transformed by the lithium
acetate method, and transformants were selected on SC plates
without uracil (SC Purification of Recombinant Progingipain--
Yeast extract was
applied to a Sepharose Q (HiTrapQ HP5, Amersham Biosciences) column
equilibrated with 20 mM Bis-Tris, pH 6.5 and washed with 5 column volumes of the same buffer following which bound protein was
eluted with a two-step gradient (0-300 mM NaCl, 20 column
volumes; and 300-500 mM NaCl, 5 column volumes). Fractions
were assayed for activity, and Western blot analysis was performed
using an enhanced chemiluminescence detection system with
anti-FLAG monoclonal antibodies (Sigma).
Fractions containing recombinant protein were pooled and further
purified by immunoaffinity chromatography by binding to the to M-2
anti-FLAG agarose gel (Sigma) slurry overnight at 4 °C. Next the gel
was washed on the column with TBS buffer (20 mM Tris, 137 mM NaCl, pH 7.6). Bound protein was eluted with
FLAG-peptide (100 µg/ml). 1-ml fractions were collected and assayed
for active enzyme or by Western blot with anti-FLAG antibodies.
Fractions containing the recombinant protein were pooled and store at
Processing of Zymogen--
Zymogen processing was analyzed in
two ways. First, purified pro-RgpB was subjected to self-processing by
incubating the recombinant protein in assay buffer (200 mM
Tris, pH 7.6, 100 mM NaCl, 5 mM CaCl2, 10 mM cysteine) for 2 h at
37 °C. Part of the reaction mixture was used for affinity labeling
and activity assay; the other part of the reaction was stopped by
adding 100 µM leupeptin, and cleavage products
were analyzed by Western blot with anti-FLAG antibodies or
antiserum to the mature RgpB (21). Second, purified pro-RgpB was tested
for self-processing at different concentrations to test for inter-
versus intramolecular activation. The RgpB antiserum was
raised against a peptide corresponding to the N-terminal 35 residues of
mature RgpB and preferentially recognizes denatured protein. It does
not require a free N terminus for reactivity as demonstrated under
"Results."
Synthesis of
Ac-biotinyl-Lys-Tyr-6-aminohexanoic-Arg-acyloxymethyl Ketone
(BiRK)--
All chemicals used in the synthesis of the
acyloxymethyl ketone were purchased from Advanced Chemtech and
Sigma. The biotinylated inhibitor BiRK was synthesized by a solid-phase
method from an arginine chloromethyl ketone according to the procedure
described in Ref. 22 with minor modifications. The chloromethyl ketone was synthesized essentially as described using Fmoc-Arg(Pbf)-OH where
Fmoc is N-(9-fluorenyl)methoxycarbonyl and Pbf is
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (23).
Following cleavage from the matrix and deprotection, BiRK was purified
on a Waters C-18 reverse phase high pressure liquid chromatography column and verified by mass spectrometry.
Determination of Inhibition Constants of RgpB by BiRK--
The
association rate of BiRK was determined by titration against RgpB of
known activity purified from P. gingivalis (24). Enzyme was
used at a concentration of 0.25 nM. Enzymatic activity was
measured by cleavage of Boc-FPR-AMC as described above. Inhibition of
RgpB with BiRK was determined by progress curve analysis as follows.
Enzyme was pre-equilibrated in assay buffer for 10 min at 37 °C and
then added to a pre-equilibrated reaction mix containing 0-10
nM BiRK and 2 mM Boc-FPR-AMC in assay buffer to
a final volume of 100 µl. Kinetic constants were obtained by a
nonlinear least-squares fit of the data to the equation:
y = (vst Active Site Labeling--
Active site labeling was performed
with a trace (non-saturated) and a high (saturated) biotinylated
affinity probe concentration. The time and concentration required to
obtain trace and saturation probe binding were estimated according to
the equation t1/2 = ln2/ka
[I] where I is the concentration of
inhibitor required for free enzyme to decrease by 50% (half-life
t1/2). A 30-µl portion of purified recombinant
proenzyme (126 ng) subjected to autoprocessing was activated in assay
buffer for 10 min at 37 °C. BiRK was added to a final concentration
of 10 nM for 5 min at room temperature (trace probe
condition) and 5 µM for 45 min at room temperature
(saturating probe condition). Active site labeling was terminated by
adding leupeptin (100 µM final concentration) for 5 min
followed by boiling samples in SDS-PAGE sample buffer.
Enzyme Activity Assay--
Routinely the activity of recombinant
forms of gingipain was determined by recording the release of
7-amino-4-methylcoumarin (AMC) generated by cleavage of Boc-QGR-AMC
(100 µM) at 37 °C by measuring the increase in
fluorescence at excitation/emission 380/490 nm using an fmax
fluorescence microplate reader (Amersham Biosciences) operating in the
kinetic mode. Assays were performed in 100 µl of assay buffer.
Specific activity was defined as the amount of AMC released/min/µg of
purified recombinant protein used for assay. The catalytic parameters
Km and kcat were calculated
by a non-linear regression fit to the Michaelis-Menten equation using
substrate at a concentration ranging from 2.3 to 300 µM.
Active enzyme concentration of fully processed recombinant wild-type
RgpB was based on active site titration with leupeptin. Because of the
low concentration of double Arg Expression of Recombinant Pro-RgpB Mutants--
Heterologous
expression of soluble and active full-length or mutant forms of RgpB in
E. coli was unsuccessful. Therefore we adapted the
constructs to a Saccharomyces expression system that is
engineered to drive synthesis and secretion. We added a FLAG epitope
tag at the C terminus of all constructs for ease of purification and
identification. Recombinant proteins were purified by a two-step protocol using anion exchange (Q-Sepharose) followed by anti-FLAG immunoaffinity chromatography and analyzed initially by Coomassie protein staining (Fig. 2A) and Western blot using a FLAG
antiserum (Fig. 2B). Yields were low and ranged from 10 to
20 µg of protein/liter of yeast culture. Overexpression of the
full-length pro-RgpB resulted predominantly in the expected 82-kDa
protein but also in partial processing demonstrated by bands of lower
molecular mass (68 and 56 kDa). The N-terminal sequence could
not be obtained for this 82-kDa protein, probably due to a blocked N
terminus, but based on the molecular mass in SDS-PAGE and
assignment of tryptic peptides by MALDI-TOF (80% sequence coverage),
the 82-kDa form corresponds to full-length zymogen with signal peptide
attached. This was unexpected since the expression system was designed
for secretion with expected signal peptide removal. The observation may
explain why the recombinant product was not released from the cells,
necessitating the extraction of the protein from the cell pellet.
N-terminal sequence analysis revealed that the 68-kDa protein began at
Ala
To assess the importance of autoprocessing we constructed individual
Arg
Native mature RgpB obtained from P. gingivalis is truncated
at the C terminus (6), and consequently we were unable to purify this
protein because the C-terminal FLAG tag would be removed. To obtain
this derivative we generated the construct pro-RgpB- Autocatalytic Processing--
Recombinant pro-RgpB proteins
were incubated at 37 °C for 2 h in assay buffer (see
"Materials and Methods") to provide conditions for autolytic
processing. After this time the full-length wild-type proenzyme was
almost completely converted to the mature form (Fig. 3, A and B). The
fully mature recombinant enzyme appeared to be identical in mass to
that of the native enzyme indicating cleavage at the same site. The
possibility that a contaminating yeast proteinase was responsible for
processing is unlikely because the general serine protease inhibitors
phenylmethanesulfonic acid (2.5 mM) and
3,4-dichloroisocoumarin (0.1 mM), the metalloprotease
inhibitor 1,10-phenanthroline (1.0 mM), and the aspartic
protease inhibitor pepstatin (0.015 mM) had no effect on
the self-processing (not shown). In contrast, the gingipain inhibitor
leupeptin at 100 µM completely prevented proenzyme
autoprocessing. Moreover, the catalytic mutant Cys244
We determined the order of processing by using differential Western
blot using antisera against the N-terminal and C-terminal (FLAG-tagged) regions. An intermediate with the N-terminal propeptide intact but lacking the C-terminal FLAG was not detected. This reveals
that removal of the C-terminal region (residues 434-506) requires
prior removal of the N-terminal pro segment. Surprisingly, conversion
to the mature form was drastically retarded in the RgpB- Activity Determination of Recombinant Progingipain Forms--
Our
data demonstrate that mature gingipain can be formed in
vitro by sequential autoprocessing with the ultimate product
lacking both the N-terminal propeptide and the C-terminal extension
exactly as seen in the active enzyme isolated from P. gingivalis. However, it is not clear whether the final product
represents the most active species or what the relative activities of
the zymogen and intermediate forms are. To address this issue and to
test the relevance of the cleavages we used active site labeling of recombinant zymogen and its derivatives. This technique relies on the
inherent ability of a single active site-directed affinity probe to
react with kinetics that parallel the enzyme catalytic competence (25).
All intermediates in the processing of pro-RgpB have a potential active
site and an intact catalytic apparatus as demonstrated by BiRK labeling
(Fig. 3, C and D), but this does not mean that
they have equivalent activities. If two species of an enzyme with
different activities are present in an equimolar amount, they will be
equally labeled only if inhibitor is in excess. Conversely, if the
inhibitor is in deficit (enzyme is saturating) the relative labeling
should reflect the activity of the forms: the most active form will be
more heavily labeled. In other words, low BiRK concentrations reflect
enzymatic activity, whereas high BiRK concentrations simply reflect
total concentration. This useful property enables us to quantitate
enzyme activity as a function of BiRK labeling.
The probe we designed, BiRK, was intended for high reactivity with
Arg-specific cysteine proteases but was broadly tolerant of extended
subsite occupancy. Labeling was performed using a low probe
concentration (10 nM) and short time of incubation (5 min)
under conditions predetermined (see "Materials and Methods") not to
saturate the potential active sites present (Fig. 3C). To
verify this procedure we also incubated the intermediates with probe
under conditions calculated to completely saturate all available active
sites (5 µM, 45 min) (Fig. 3D). The intensity
of the bands labeled at high probe concentration was comparable to the
intensity of bands probed with antibodies, reflecting the total protein of each form (Fig. 3, B and D, compare
lanes 1). In stark contrast, low probe concentrations
revealed a completely different pattern with the fully processed
enzyme, even though it is present in the lowest amount, displaying
highest labeling (Fig. 3, B and C, compare
lanes 1). Full-length zymogen, representing the highest concentration among the proteins, showed the lowest labeling under low
probe conditions. These findings indicate that the enzyme gains
activity with each of the three processing events.
The same samples were also analyzed for enzymatic activity. When
incubating with the fluorometric substrate Boc-QGR-AMC a 3-fold
increase in substrate cleavage was detected in wild-type digest, and no
increase in activity was observed for C-terminally truncated enzyme and
double mutant (Fig. 6). Taken together, the data argue that pro-RgpB
activation correlates with the enzyme processing and is accomplished by
sequential cleavage of N-terminal propeptide followed by trimming of
the C terminus.
Processing of Pro-RgpB Is a Bimolecular Process--
Zymogen
processing could be either intramolecular (by the catalytic site within
the precursor) or intermolecular (where a different catalytic site
attacks the bonds in an adjacent molecule). These possibilities can be
distinguished by determining whether the process is unimolecular or
bimolecular (26). The rate of autoprocessing was dependent on precursor
concentration (Fig. 7), implying a second
order process. Since this is consistent with a bimolecular mechanism,
we conclude that processing is predominantly intermolecular, although
we cannot rule out some intramolecular component.
Catalytic Properties of Recombinant Gingipain Forms--
Probe
labeling is consistent with an increase in activity during processing,
and we attempted to confirm this by determining the kinetic parameters
of full-length wild-type protein converted to the mature form by
self-processing and full-length double mutant (Arg
Based on the estimated
kcat/Km values (Table
I) we observed an approximately 80-fold
increase in catalytic efficiency during conversion of zymogen to the
mature enzyme. Significantly the kinetic parameters of fully processed
wild-type pro-RgpB are close to that of the native enzyme isolated from
P. gingivalis.
The initial translation product deduced from the rgpB
gene is a precursor protein with a 24-residue signal peptide,
229-residue N-terminal pro region, and a 72-residue C-terminal region
that are not found in the mature native enzyme. Although previously postulated (27, 28), the presence of an RgpB precursor has not
previously been demonstrated in P. gingivalis, and
production of recombinant active gingipains in E. coli with
activity comparable to the natural enzymes has been unsuccessful (29,
30). The latent activity of almost all proteases is usually restrained by embedding them in a precursor that must be processed to generate the
active form, thus protecting biosynthetic machinery and allowing for
activation control (12). This study aimed to produce the precursor form
of RgpB with a view to understanding the reason for the precursor: was
it to secure the zymogen or was it required for folding of the
catalytic form? Because RgpB is naturally a secreted product we
reasoned that a heterologous secretion/expression system would most
faithfully simulate the natural folding and processing events, hence
our choice of a yeast expression system.
We provide direct evidence that recombinant pro-RgpB can be
autocatalytically processed to generate a mature form of the enzyme equivalent to that isolated from P. gingivalis culture
supernatants. Maturation of pro-RgpB occurs through the sequential
appearance of intermediates leading to the ultimate product (Fig.
8). The first cleavage at
Arg
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
alg6::HIS3,
ade2-101, his3
200,
ura3-52, lys2-801) was kindly provided by
Markus Aebi (Institute of Microbiology, ETH Zentrum, Zurich). Escherichia coli DH5
was used as the host for
construction and propagation of all plasmids. Standard and synthetic
media were prepared and supplemented with nutrients appropriate for
selection and maintenance of plasmids as described previously (16).
Yeast cells were grown in 2% glucose as carbon source and 2%
galactose and 1% raffinose to induce protein expression from
GAL1 promoter.
Acc65I)
expression vector was produced for internal laboratory purposes as
follows, although the deletion of Acc65I was not
specifically necessary for the cloning strategy used in this report.
The pRS316(
Acc65I) was first generated by digestion of pRS316 (17) with Acc65I and blunt ending of the overhang by T4 polymerase and religation in the presence of
Acc65I. Clones lacking the KpnI/Acc65I
site were identified by restriction enzyme digest and used to generate
the expression vector. The sequence encoding the Gal1
promoter was amplified from genomic yeast DNA using primers
Gal1f (5-ttggagctcacatggcattaccaccatatacatatc-3) and Gal1r
(5-agagcggccgccggtaccgttttttctccttgacgttaaagt-3). This fragment was
introduced intro pRS316(
Acc65I) as a
SacI-NotI insert resulting in pRS316Gal plasmid.
The full-length pro form of RgpB was amplified from genomic DNA
isolated from P. gingivalis strain HG66. The 24-residue
signal peptide of gingipain was replaced by the signal peptide of yeast
carboxypeptidase Y amplified from pRA21 plasmid (18). The insert was
then ligated into the yeast expression plasmid pRS316Gal, and insertion
of a FLAG epitope sequence (DYKDDDDK) at the C terminus resulted
in the pro-RgpB FL1-WT
construct (Fig. 1). The XhoI restriction site encoding two amino acids (Leu and Glu) separates the FLAG tag sequence from the authentic C-terminal sequence of pro-RgpB. Specific mutants (Fig.
1) were constructed using an overlap polymerase chain reaction technique and the pro-RgpB FL-WT constructs as a template. The double
mutant2 Arg
1
Ala/Arg
103
Ala was generated in the same way but
with pro-RgpB FL Arg
103
Ala as a template. All
constructs were sequenced completely to confirm that no undesired
mutations were present.
uracil) for auxotrophic selection (19).
Large scale expression was performed as follows. Single colony
transformants were used to inoculate 20 ml of SC
uracil medium,
and cultures were grown at 30 °C for 24 h followed by
inoculation to 1.0 liter of medium, and the culture was allowed
to grow for 12 more h. Next cells were washed in water and inoculated
into induction medium (SC
uracil, 2% galactose, 1%
raffinose). Cells were harvested by centrifugation 6 h after induction, washed in water, and resuspended in 20 mM
Bis-Tris, 10 mM NaCl, 1 mM CaCl2,
pH 6.5 containing the protease inhibitors 1 mM
phenylmethanesulfonic acid and 100 µM
3,4-dichloroisocoumarin (20). A half-volume of glass beads was added,
and the cells were mechanically broken in a bead beater. Cell debris
were removed by centrifugation at 18,000 × g for 30 min followed by filtration, and the supernatant fluid was used for
further purification.
70 °C. Identification of the recombinant protein was achieved by using both N-terminal sequencing and mass spectrometry (MALDI-TOF).
(vs
v0)(1
exp
kt)/kobs)
A. The slope of the plot of kobs
versus inhibitor concentration was used to determine the
second order rate constant ka = 3.5 × 105 M
1s
1.
1
Ala/Arg
103
Ala pro-RgpB mutant and its relatively
poor activity, we were not able to accurately evaluate the enzyme
concentration by active site titration. Therefore we estimated the
enzyme concentration based on protein concentration determined
by a modified Bradford assay (Pierce) with adjustment for the active
pro-RgpB using incorporation of BiRK.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
104, while the 56-kDa protein began at
Tyr1 (see Fig. 1 for a
description of the numbering system). Since both of these residues
follow Arg in the coding sequence, it is likely that they are generated
by a self-processing mechanism. To clarify whether the conversion
occurs autocatalytically or whether it is due to a yeast-encoded
protease we expressed a catalytic mutant, Cys244
Ala.
Substitution of Ala for the catalytic Cys completely abolished the
smaller products, confirming an autoprocessing mechanism (Fig. 2, A and B).
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Fig. 1.
RgpB constructs expressed in this study.
The initial transcript of the rgpB gene consists of an
N-terminal propeptide and a C-terminal extension that flank the mature
enzyme. The propeptide cleavage sites are denoted by
arrowheads, and the catalytic Cys residue is denoted by a
star. For purification and identification purposes all
constructs were tagged at the C terminus with the FLAG sequence
(DYKDDDDK). The numbering system used throughout this report places
residue 1 at the N terminus of the mature enzyme from the crystal
structure (6) with residues upstream of this in the precursor preceded
by a negative sign (44). The lower panel illustrates the
limits and relative expression levels of the various constructs.
R-1A, Arg 1
Ala; R-103A,
Arg
103
Ala.
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Fig. 2.
Recombinant pro-RgpB expression in
yeast. Recombinant proteins were purified from yeast cells
extracts. A, Coomassie protein staining of full-length
wild-type zymogen (FL-WT), C-terminally truncated zymogen
( C-WT), and mutants described in Fig. 1 based on a
full-length background. B, Western blot probed with
anti-FLAG antiserum of equivalent samples to those in panel
A and including individual Arg
1
Ala
(R-1A) and Arg
103
Ala (R-103A)
mutants. The intermediate band denoted
103 represents the
truncation at the Arg
103, and
1 denotes the
protein with the same N terminus as the native RgpB as
identified by N-terminal sequencing. MW, molecular mass
markers.
Ala substitutions at the determined cleavage sites and a
double mutant containing both substitutions. Both pro-RgpB Arg
103
Ala and the double mutant were found to be
unprocessed, whereas an intermediate 68-kDa form corresponding to
pro-RgpB processed at Arg
103 was detected in the
Arg
1
Ala mutant (Fig. 2B). This indicates
that pro-RgpB undergoes sequential two-step processing in which
cleavage at Arg
103 is required for subsequent processing
at Arg
1.
C-WT that
encodes the pro form lacking the 72-residue C-terminal sequence (Fig.
1) Expression and purification of this protein gave a band pattern
similar to that of a full-length protein, but as expected, each
derivative was slightly smaller (Fig. 2B).
Ala incubated in the same conditions remained unprocessed (Fig. 4A). The double mutant
(Arg
1
Ala/Arg
103
Ala)
subjected to self-processing did not generate any bands recognized by
antisera (Fig. 5, A and
B). However, affinity labeling revealed additional bands
that resulted from an aberrant cleavage (Fig. 5, C and
D). The aberrant product did not accumulate during self-processing conditions, and we did not observe an increase in
activity as determined by cleavage of Boc-QGR-AMC (Fig.
6) or affinity labeling (Fig. 5,
C and D).
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Fig. 3.
Self-processing/activation of pro-RgpB.
Purified recombinant full-length wild-type zymogen was incubated for
2 h at 37 °C, and the reaction products were visualized by
Western blot or affinity labeling with BiRK. A, proteins
probed with anti-FLAG; B, proteins probed with specific
anti-N-terminal RgpB antiserum; C, proteins labeled with a
trace concentration of BiRK; D, proteins labeled with a
saturating amount of BiRK. In each panel lane 1 represents
untreated protein, and lane 2 represents incubated protein.
The different forms of the recombinant enzymes are indicated to the
right. E, structure of the affinity label BiRK.
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Fig. 4.
Self-processing of pro-RgpB mutants.
Pro-RgpB mutants were subjected to self-processing conditions and
analyzed by Western blot using anti-FLAG and anti-N-terminal RgpB
antiserum. A, full-length catalytic mutant; B,
C-WT mutant. In each panel lane 1 represents untreated
protein, and lane 2 represents incubated gingipain.
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Fig. 5.
Self-processing of pro-RgpB double
mutant. Purified recombinant Arg 1
Ala/Arg
103
Ala mutant was incubated for 2 h at
37 °C, and the reaction products were visualized by Western blot or
affinity labeling with BiRK. A, proteins probed with
anti-FLAG; B, proteins probed with specific anti-N-terminal
RgpB antiserum; C, proteins labeled with a trace
concentration of BiRK; D, proteins labeled with a saturating
amount of BiRK. In each panel lane 1 represents untreated
protein, and lane 2 represents incubated protein. Aberrant
products are highlighted by an asterisk.
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Fig. 6.
Activation of pro-RgpB by
self-processing. The indicated recombinant pro-RgpB forms were
subjected to self-processing by incubating for 2 h at 37 °C.
Activity was measured by Boc-QGR-AMC substrate hydrolysis. The
untreated samples (white bars) were stored on ice for the
same period of time as the treated ones (black bars) and
incubated for 10 min at 37 °C in the assay buffer prior to assay.
R-103A/R-1A, Arg 1
Ala/Arg
103
Ala.
C-WT mutant
zymogen exposed to autoprocessing conditions, indicating the importance
of the C-terminal part of the protein in maturation (Fig.
4B). The RgpB-
C-WT must be relatively inactive because it
does not autoprocess in vitro, although conditions in yeast
must have been more favorable to produce the initial cleavage events.
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Fig. 7.
Effect of proenzyme concentration on in
vitro autoprocessing. Varying amounts of purified
recombinant wild-type pro-RgpB were incubated for 30 or 120 min in
assay buffer at 37 °C. The samples were treated with 100 µM leupeptin to terminate processing, and an equal amount
of protein was analyzed by Western blot using specific anti-N-terminal
RgpB antiserum. Input volume refers to the volume of
pro-RgpB (12 µg/ml) added to the 40-µl total reaction volume.
1
Ala/Arg
103
Ala), which represents the zymogen form.
Labeling with BiRK revealed the full-length zymogen and an aberrant
product of about 45 kDa (Fig. 5, C and D). The
latter probably corresponds to a cleavage C-terminal to
Arg
1 since it is not recognized by the specific anti-RgpB
antiserum raised to the first 35 residues of mature RgpB. The aberrant
cleavage appears to be a result of mutating Arg
103 and
Arg
1 and must be taken into account for quantitating
activity due to the full-length zymogen. Quantitative image analysis
revealed that the full-length zymogen corresponds to 66% of the total
RgpB as determined under saturating BiRK conditions (Fig.
5D). Thus we estimated the protein concentration to be 66%
of that determined by a dye binding assay (see "Materials and
Methods"). The full-length form corresponds to 55% of activity as
determined under trace BiRK conditions (Fig. 5C). Therefore
we estimated that 55% of the velocity against Boc-QGR-AMC is due to
the full-length zymogen. This is based on the assumption that probe
labeling efficiency parallels catalytic efficiency. Therefore these
values must be taken into account for an accurate estimate of enzyme
concentration and estimate of
kcat/Km.
Kinetic parameters of recombinant mature RgpB and zymogen mutant form
(Arg1
Ala/Arg
103
Ala)
1
Ala/Arg
103
Ala) cannot be converted, thus this form
represents the full-length zymogen. The parameters describe the
respective properties with Boc-QGR-AMC as the substrate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
103 is essential for further processing since mutation
at this site abolishes generation of authentic downstream
intermediates. The second processing step removes the remainder of the
N-terminal propeptide, and the final step yields the mature enzyme by
removal of a C-terminal peptide. Unfortunately we were not able to
identify the cleavage site at the C terminus because of the difficulty in obtaining the peptide. Nevertheless, conversion of pro-RgpB into the
mature enzyme can be attributed to the action of the enzyme itself. The
conversion can take place during expression and/or purification but
also in vitro with the purified components. Naturally other
bacterial factors may influence processing in vivo,
but our data demonstrate that an inherent processing pathway exists.
This implies that the precursor must have at least a small degree of
proteolytic activity, hence our attempts to measure the relative
activity of the various intermediates.
View larger version (33K):
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Fig. 8.
Proposed model for RgpB activation. In
the schematic the gray ovals represent the N- and
C-terminal extensions not found in the fully processed enzyme. The
first phase in activation is intermolecular removal of the N-terminal
propeptide (pro), which occurs in two steps. This is
followed by a second phase during which the C-terminal extension
(C) is removed. According to the crystal structure (6) the
catalytic domain is composed of a core with catalytic sites
(cat.) and a structure that stabilizes the conformation of
the active form (cap). The active domain sits on top of an
Ig domain that has intimate contacts with both the catalytic site and
the active form. We propose that autolytic processing removes
constraints to promote active site formation through conformational
coupling of the three main structural units.
It proved impossible for us to obtain full-length wild-type pro-RgpB
because of processing in yeast, and therefore we resorted to obtaining
material mutated at the 1 and
103 processing sites, which we define
as the "frozen zymogen." This full-length material was somewhat
active with an apparent
kcat/Km value 80-fold lower
than that of the mature protein. It is not unusual for protease zymogens to have proteolytic activity, but normally this activity is
many orders of magnitude lower than that of the active enzyme (12).
There are a few examples of protease precursors with substantial activity compared with its processed product, including tissue-type plasminogen activator (31), coagulation factor VII (32), and caspase-9
(13, 33). However, in each of these cases intrinsic activity of the
processed form is low and requires enhancement by cofactors. This
appears not to be the case with pro-RgpB since the frozen zymogen form
has considerable catalytic activity with an estimated
kcat/Km of 1.4 × 104 M
1s
1, higher
than some fully processed proteases on equivalent synthetic substrates.
Interestingly the activity of the enzyme seemed to increase with each
processing step as demonstrated by an enhanced interaction with the
affinity probe (Fig. 3C). Although it is difficult to
quantitate the probe data, the increase in probe labeling is consistent
with an 80-fold increase in catalysis.
Conceivably the 80-fold lower activity of the zymogen may reflect the need to restrain activity during protein synthesis and delivery in P. gingivalis, but there is another plausible explanation for the existence of the precursor. We were not able to obtain protein from a construct encoding the mature protein. This indicates that the propeptide may serve an intramolecular chaperone function analogous to the propeptide of subtilisin-like proteases. Extensive work on subtilisin demonstrates that the 77-residue subtilisin propeptide is not required for enzymatic activity and is removed intramolecularly by autoprocessing upon the completion of the protein folding (for a review, see Ref. 34). This intramolecular chaperone function for protease N-terminal propeptides is not restricted to subtilisins but also encompasses other families (35, 36). Significantly the intramolecular chaperone propeptides frequently also act as potent inhibitors of the enzymes, decreasing enzyme activity several orders of magnitude (37). Since the RgpB propeptide seems to decrease activity 80-fold it may function as an inhibitor, but it is more plausible that its role is to serve in the folding of the catalytic domain of RgpB. In support of this is the inability of our group and several other groups to express the catalytic domain (residues 1-506) in an active or soluble form (29, 30).
Perhaps the greatest challenge is to relate the structure/function of
the RgpB propeptide to the activation mechanism of its cousins,
separase, the legumains, and caspases. On the basis of conservation of
mechanism one would expect the fundamental activation mechanisms to be
essentially identical. Both yeast and human separase undergo autolytic
processing, yet this does not seem to be responsible for activation of
the enzymes (38, 39). Separase may require a separate
chaperone/inhibitor known as securin to allow correct folding and
generation of a latent active site. In contrast, human legumain has
been proposed to undergo an activating C-terminal cleavage (40) similar
to the final activating cleavage demonstrated above for RgpB. On the
other hand, recent x-ray structures of zymogen forms of caspase-7 (14,
15) and caspase-9 (13) demonstrate a fundamentally different mechanism.
In both cases the zymogens show at least 3 orders of magnitude less
activity than that of the fully active forms (33, 41), and this is
caused by a dislocation of two loops that contain the major activity
and specificity determinants of each protease. Although the driving
forces are distinct (cofactor binding for caspase-9 and proteolysis for
caspase-7) activation results from reordering of these two loops. Like
RgpB, the precursors of caspases retain a reduced ability to
incorporate affinity probes (25, 42, 43), which is not typical of most
protease zymogens, and we take this as preliminary evidence that
pro-RgpB is activated in a similar manner (Fig. 8). This would mean
that the propeptide may not block access of substrate to a fully formed
active site as seen in many protease families. More likely the
propeptide docks with the region between the catalytic domain and cap
domain thereby restricting ordering of the activation loop (Fig. 8). Proteolytic removal would then allow activation by a caspase-like mechanism, but further speculation should await structural evidence.
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ACKNOWLEDGEMENT |
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We thank Scott Snipas for outstanding technical support.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA69381 (to G. S. S.), California Breast Cancer Research Program Fellowship 8GB-0137 (to K. M. B.), and National Institutes of Health Grant DE009761 and Committee of Scientific Research, Poland Grant KBN-6 P04A 047 17 (to J. P.).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: The Burnham Inst., 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3114; Fax: 858-713-6274; E-mail: gsalvesen@burnham.org.
Published, JBC Papers in Press, January 17, 2003, DOI 10.1074/jbc.M210564200
2 The minus sign before the number indicates residues in the RgpB prodomain counting backward using the first residue of the mature protein as the origin.
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ABBREVIATIONS |
---|
The abbreviations used are: FL, full-length; WT, wild-type; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; BiRK, Ac-biotinyl-Lys-Tyr-6-aminohexanoic-Arg-acyloxymethyl ketone; Boc, t-butoxycarbonyl; AMC, 7-amino-4-methylcoumarin; SC, synthetic complete.
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