From the Departments of Biochemistry,
§ Microbiology and Immunology, ¶ Medical
Technology, and
Pediatrics, National Cheng Kung University
College of Medicine, 1 University Road, Tainan 701, Taiwan
Received for publication, September 4, 2002, and in revised form, February 14, 2003
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ABSTRACT |
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Streptopain is a cysteine protease
expressed by Streptococcus pyogenes. To study the
maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R,
N356D and W357A mutant proteins were expressed in Escherichia
coli and purified to homogeneity. Proteolytic analyses showed
that the maturation of prostreptococcal pyrogenic exotoxin B zymogen
(pro-SPE B) involves eight intermediates with a combination of
cis- and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The
relative autocatalytic rates of these mutants exhibited the order
Q186N > W357A > N356D, C192S, H340R. Interestingly, the
N356D mutant containing protease activity could not be converted into
the 28-kDa form by autoprocessing. This observation suggested that
Asn356 might involve the cis-processing of the
propeptide. In addition, the maturation rates of pro-SPE B with trypsin
and plasmin were 10- and 60-fold slower than that with active mature
streptopain. These findings indicate that active mature streptopain
likely plays the most important role in the maturation of pro-SPE B
under physiological conditions.
Group A streptococcus
(GAS)1 causes several human
diseases, including as pharyngitis, acute rheumatic fever, scarlet
fever, post-streptococcal glomerulonephritis, and toxic shock-like
syndrome (1, 2). Virtually all strains of GAS isolated from patients with invasive disease express an extracellular cysteine protease known
as streptopain (EC 3.4.22.10), with synonyms including streptococcal
pyrogenic exotoxin B (SPE B or SpeB), streptococcus peptidase A, and
streptococcal cysteine protease (3-5). Many reports also suggest that
streptopain is an important virulence factor in streptococcal
infections (6-11). Streptopain produced from GAS is released
extracellularly to culture medium as a zymogen (pro-SPE B) with a
molecular mass of 40-kDa, and the active form is a 28-kDa mature
protease (12-16). Because the 28-kDa active form of streptopain plays
an important role in GAS pathogenesis, studies on the maturation of
pro-SPE B have become a subject of interest (12, 14, 15,
17).
Zymogen activation produces a prompt and irreversible response to a
physiological stimulus, and it is capable of initiating new
physiological functions. The maturation of a zymogen involves limited
proteolysis and can be finished either in a single activation step or
in a consecutive cascade. The maturation of pro-SPE B can be achieved
under a variety of conditions, including proteolysis by autoprocessing,
trypsin, subtilisin, or the 28-kDa active form of streptopain (12, 14,
15, 17). Similar autoactivation processes have been found in other
families of cysteine proteases; specifically, the maturation mechanism
of propapain has been fully characterized (17-20). The
cis-processing of propapain is characterized by a zero-order
reaction, where the rate of propapain processing is independent of the
propapain concentration. In contrast, the trans-processing
of propapain is characterized by a higher order reaction, where
the rate of propapain processing is dependent on the concentration of
propapain (20). In one recent study, investigators used multiple
approaches to demonstrate that the maturation of pro-SPE B involves
both cis- and trans-processing (17). Pro-SPE B
immobilized on a Sepharose resin is capable of liberating the 28-kDa
form of streptopain from the column, indicating that the maturation of
pro-SPE B involves cis-processing (17).
Although streptopain (C10) belongs to one of the cysteine
protease families and undergoes similar maturation processing, it contains a prodomain different from that of other cysteine protease families (20, 21). The maturation of pro-SPE B to the 28-kDa active
form of streptopain can also be mediated by exogenous proteases such
trypsin and subtilisin and by the active form of streptopain (14).
Streptopain is an extracellular plasmin-binding protein for
nephritogenic streptococci (22). The ability of streptopain to bind
human plasminogen and plasmin may be important because these
interactions may provide a means for GAS invasion. However, the
maturation of pro-SPE B by exogenous proteases has never been fully addressed.
To date, >60 families of proteases containing a cysteine residue at
the active site have been found (21). According to the MEROPS Protease
Database,2 streptopain and
papain belong to the C10 and C1 families of
papain-like clan CA, respectively. Although there is only 20% identity
and 43% similarity between the sequences of streptopain and papain, the catalytic residues of streptopain (Cys192,
His340, and Asn356) occur in the same order as
those in papain (Cys25, His159, and
Asn175), with some identical nearby residues (5, 20).
However, little is known about the residues involved in the maturation processing of streptopain. For purposes of comparison with previous mutagenesis studies, in this study, we numbered the first methionine of
the signal peptide of streptopain as position 1 (23-25).
To examine the kinetic and biochemical effects of mutation on the
maturation processing of pro-SPE B, in this study, we expressed the
wild-type protein and mutants Q186N, C192S, H340R, N356D, and W357A in
Escherichia coli: Gln186 is the residue outside
the substrate-binding pocket and contacts with the loop containing
Cys192; Cys192 and His340 are the
catalytic residues; and Asn356 and Trp357 are
the residues near the active site of streptopain. Studies of the
chemical modifications of cysteine, histidine, and tryptophan also
indicate that these residues are essential for the protease activity of
streptopain (5). Based on the x-ray structure of the streptopain C192S
mutant (Fig. 1) (27), the catalytic site of streptopain has a catalytic Cys-His dyad, which differs from most
other cysteine proteases containing a Cys-His-Asn catalytic triad.
Previous studies have shown that a mutation at Cys192 or
His340 of streptopain leads to a complete loss of protease
activity (4, 23, 25). Asn175 of papain is not essential for
its protease activity, and it may play a role in orientating the
catalytic residues. In streptopain, the carbonyl group of
Trp357 may replace the function of Asn175 in
papain (26). The crystal structure also demonstrates that Phe342, Trp357, and Phe367 form a
hydrophobic pocket that fits a hydrophobic residue at the P2 site of
the substrate (26). Although the role of Asn356 in
streptopain is not clear, it is near the active-site region of
streptopain. To compare the maturation of pro-SPE B and propapain, we
applied the same approach used in propapain to study pro-SPE B (20). We
also investigated the processing of limited proteolysis of pro-SPE B by
trypsin and plasmin and characterized the intermediates of pro-SPE B
during its course of maturation. Our goal was to provide an additional
basis for exploring the differences between autoproteolytic and
proteolytic processing. We found that the maturation processing of
pro-SPE B involves both cis- and trans-processing in a consecutive cascade with eight identifiable intermediates and that
the 28-kDa active form of streptopain is the most effective protease
for converting pro-SPE B into a mature protease through trans-processing. This study serves as the basis for gaining
insight into streptococcal infections by exploring the maturation
processing and characterization of streptopain.
Streptopain Expression, Mutant Construction, and
Purification--
The genomic DNA of GAS was extracted from strain
A20. The structural gene of pro-SPE B was amplified by PCR using the
sense primer
5'-GGATCCGGATCCCATCATCATCATCATCATGATCAAAACTTTGCTCGTAACGAA-3' with a His6 tag and BamHI recognition and by the
antisense primer 5'-GGATCCGGATCCCTAAGGTTTGATGCCTACAACAG-3'
with BamHI recognition. The PCR product was purified
and then cloned into the BamHI site of the pET-21a vector.
The recombinant plasmid was transformed into the E. coli BL21(DE3) pLys strain, and the system was under the
control of a strong T7 promoter. The wild-type construct was used to
produce Q186N, C192S, H340R, N356D, and W357A mutations using overlap
extension PCR (27). Cells were grown at 37 °C for 6-8 h in LB
medium (1 liter of 10 g of Bacto-Tryptone, 5 g of Bacto-yeast
extract, and 10 g of NaCl) that was adjusted to pH 7.2 with 3 N NaOH. The cells were cultured to
A600 = 0.5-1.0. To the culture was added
isopropyl-1-thio-
To obtain soluble proteins, the conditions of protein expression were
optimized by lowering the temperature and by varying induction periods.
Under varying induction conditions, 10 ml of cells were collected by
centrifugation and suspended in 1 ml of lysis buffer (20 mM
Tris-HCl, 200 mM NaCl, and 1 mg/ml lysozyme, pH 8.0). Each
lysate was centrifuged at 10,000 × g for 10 min. The
proteins in the supernatant (soluble) were collected, and the proteins
in the pellet (insoluble) were dissolved in 1 ml of sample buffer.
SDS-PAGE was performed to analyze the relative proportions of
overexpressed proteins in the soluble and insoluble fractions.
Recombinant wild-type streptopain was converted into a 28-kDa active
enzyme during the course of purification. To prevent the conversion,
mercuric chloride was added to the wild-type extract to a final
concentration of 1 mM and was kept present throughout the
purification. In addition, most of the expressed W357A mutant was
present as an insoluble inclusion body, and a standard procedure using
a denaturing condition was performed to refold the protein (41). The
inclusion body of the W357A mutant was solubilized in denaturing
solution (4.5 M urea, 20 mM Tris-HCl, and 200 mM NaCl, pH 8.0), and the solution was diluted to
A280 < 0.1. The protein was renatured by
dialysis against 20 mM Tris-HCl and 200 mM
NaCl, pH 8.0. The recombinant proteins were purified by
Ni2+ chelating chromatography (Amersham Biosciences) with a
gradient of 20-200 mM imidazole. The proteins were
concentrated by Amicon ultrafiltration using a 10-kDa cutoff membrane
and then exchanged with PBS. The final solutions were stored at
Purification of Streptopain from Streptococcus Strain
A20--
Native streptopain was purified from strain A20 as previously
described (8). One volume of 1 ml of bacterial culture was first grown
overnight at 35 °C in 20 ml of TSBY medium (3% tryptic soy broth
and 0.5% yeast extract). The culture was then added to 100 ml of TSBY
medium. Streptopain was produced by growing cells at 37 °C for
22-24 h. The supernatant was collected by centrifugation and filtered
through a 0.45-µm membrane filter. The filtrate was diluted with 400 ml of cold distilled water, and the pH was adjusted with 1 N NaOH to 8.0. Then, 25 g of pre-equilibrated
DEAE-Sepharose resin (Amersham Biosciences) with 20 mM
Tris-HCl, pH 8.0, were added to the filtrate. The solution was left for
30 min with occasional mixing, and the unbound protein was collected by
filtration. The filtrate was concentrated to 100 ml by Amicon
ultrafiltration using a 3-kDa cutoff membrane. The buffer was exchanged
by ultrafiltration with 1 liter of 20% ethanol and 20 mM
Tris-HCl, pH 7.0. The final solution was loaded onto a Red A
column (Dymatrex gel, Millipore Corp.). Streptopain was eluted using a
linear gradient of 400 ml of 0-2 M NaCl with a flow rate
of 20 ml/h, and 5-ml fractions were collected. SDS-PAGE showed that
streptopain with a molecular mass of 28 kDa was homogeneous.
Azocasein Assay--
The azocasein assay was used to test for
proteolytic activity of streptopain and mutant proteins. The assay was
modified as previously described (28). Activity was determined by
measuring the hydrolysis of azocasein based on the absorbance increase
at 366 nm against time as described below. The reaction was initiated by addition of 20 µl of streptopain or mutant protein to 160 µl of
reaction mixture containing 2.7 mg/ml azocasein, 5 mM
dithiothreitol (DTT), and 5 mM EDTA (Sigma) in PBS. After
incubating the solution at 37 °C for the designated time intervals
ranging from 0 to 24 h, the reaction was stopped by addition of 40 µl of 15% ice-cold trichloroacetic acid. Absorbance was measured
using a Beckman Model DU640 spectrophotometer. One enzyme unit is
defined as the amount of protease required to release 1 µg of soluble
azopeptide/min. The specific absorption coefficient
(A Processing of the Pro-SPE B C192S Mutant by Wild-type and Mutant
Proteins--
Because the pro-SPE B C192S mutant does not
exhibit any enzyme activity and exists as a 42-kDa zymogen, it can be
used as the substrate for the 28-kDa active form of streptopain (17). The reaction was carried out in a total of 20 µl of PBS containing 5 mM EDTA and 5 mM DTT. Purified or recombinant
streptopain protein at a final concentration of 1.2 µM
was incubated with the 42-kDa C192S mutant (24 µM) at
37 °C for the designated time intervals ranging from 0 to 14 h.
The reactions were quenched by addition of 5 µl of 50 µM E-64
(trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane) and incubated at 37 °C for 30 min. The solution was then heated at
100 °C for 10 min and analyzed by 12% SDS-PAGE. Gels were scanned using a Vilber Lourmat Model CN-TFX imaging system, and the intensities of the bands were integrated with BIO-1D Version 5.07 software. Proteolytic activity was measured as the disappearance of pro-SPE B,
and relative reaction rates were obtained from the intensity change of
the 42-kDa pro-SPE B C192S band against time.
Processing of the Pro-SPE B C192S Mutant by Plasmin and
Trypsin--
Plasmin and trypsin were obtained from Sigma. The
reaction was carried out in a total of 20 µl of PBS containing 5 mM EDTA and 5 mM DTT. Plasmin or trypsin at a
final concentration of 1.2 µM was incubated with the
42-kDa pro-SPE B C192S mutant (24 µM) at 37 °C for the
designated time intervals ranging from 0 to 72 h. The reactions
were quenched by addition of 5 µl of 5 mM
phenylmethanesulfonyl fluoride and incubated at 37 °C for 30 min.
The solution was then heated at 100 °C for 10 min and analyzed by
12% SDS-PAGE. The relative rates of processing were measured from the
intensity change of the 42-kDa pro-SPE B C192S band against time.
Autocatalysis Assay--
The reaction was carried out in PBS
with 5 mM EDTA and 5 mM DTT. A total of 13.5 µg of pro-SPE B were incubated in volumes of 20, 35, 50, 75, 100, 200, 300, and 400 µl at 37 °C for the designated time intervals
from 0 to 14 h. The final concentrations were 0.2, 0.35, 0.5, 0.75, 1, 2, 3, and 4 µM, respectively. The reaction was
quenched by adding 5 µl of 50 µM E-64 and heating the
solution at 100 °C for 10 min, and the solution was then lyophilized for gel analysis. Relative reaction rates were obtained from the intensity change of pro-SPE B against time.
Mass Spectrometric Measurement--
The molecular masses of the
proteins were examined by mass spectrometry using a PerkinElmer
Life Sciences triple quadrupole mass spectrometer (Model API365).
Proteins were dissolved in 0.1% formic acid and 100% methanol as the matrix.
N-terminal Sequence Analysis--
Streptopain, mutant proteins,
and the cleavage products of wild-type and mutant proteins of pro-SPE B
were analyzed by 12% SDS-PAGE and transferred to a polyvinylidene
difluoride membrane. The membrane was stained with 0.2% Amido Black.
The bands were excised and analyzed using an Applied Biosystems Model
477A Sequencer.
CD Spectroscopy--
CD spectroscopy was used to determine the
secondary structures of wild-type and mutant proteins of pro-SPE B. CD
spectra were measured at 27 °C on a Jasco J-720 spectropolarimeter
that had been calibrated with camphosulfonic acid. Spectra were
recorded under nitrogen between 185 and 260 nm and with a 1.0-nm
spectral step size, a 1.0-nm bandwidth, and a 10 or 12 nm/min scan
rate. The secondary structures of streptopain and its mutants were
estimated using the convex constraint algorithm together with a
least-square fitting program (LINCOMB) as described by Perczel et
al. (29, 30).
Expression and Purification--
Wild-type pro-SPE B and mutants
Q186N, C192S, H340R, N356D, and W357A without their signal peptides
were expressed in an E. coli pET-21a expression system. All
recombinant proteins contained 11 extra vector residues (ASMTGGQQMGS)
and 6 histidine residues to simplify the purification procedures. All
recombinant proteins were purified to apparent homogeneity in a single
step by Ni2+ chelating chromatography. Recombinant
wild-type streptopain was expressed as 42-kDa pro-SPE B and converted
into a 28-kDa active enzyme during the course of purification. To
prepare 42-kDa pro-SPE B, 1 mM HgCl2 was added
as an inhibitor to prevent the conversion. In contrast, all mutant
proteins were purified as 42-kDa zymogens without adding
HgCl2 as an inhibitor. However, they formed inclusion bodies when the cells were induced at 37 °C. To obtain
soluble protein and to maximize the yield, the cells were grown at
different temperatures and induction periods. We determined the
relative proportions of overexpressed proteins in the soluble and
insoluble fractions that were obtained by varying the induction
conditions. We found that 100, 68, 42, and 5% of the C192S mutant
protein were soluble when the C192S mutant cells were induced at
28, 30, 32, and 37 °C, respectively. Similar approaches were used to
obtain 100% soluble proteins for other mutants. The optimal induction temperatures for the cells of H340R, N356D, and W357A mutants were 28, 25, and 17 °C, respectively. The final yields of the purified
wild-type and Q186N, C192S, H340R, N356D, and W357A
mutant proteins were ~45, 40, 380, 210, 15, and 8 mg/liter, respectively (Table I). Based
on SDS-PAGE analysis, the wild-type and mutant proteins were
homogeneous (Fig. 2). Wild-type streptopain was freshly prepared to prevent
autodegradation. The mutant proteins remained stable as 42-kDa zymogens
at Structural Studies of Wild-type and Mutant Streptopain by CD
Spectroscopy--
To examine the structural differences between native
and recombinant streptopain while excluding mutation effects due to
conformational changes, we performed CD analyses to determine the
secondary structures of wild-type and mutant streptopain. The CD
spectra were fit with a root mean square deviation of 2-5% using the
convex constraint algorithm together with the least-square fitting
program (LINCOMB) of Perczel et al. (29, 30). Although a
slight difference at ~210 nm was observed, CD analysis showed that
recombinant and native 28-kDa streptopain had 18.8 and 20.2%
Proteolytic Activities of Wild-type and Mutant
Streptopain--
We used azocasein as the substrate to examine the
proteolytic activities of the wild-type and mutant proteins of
streptopain. As shown in Table I, mutants C192S and H340R of SPE B
completely lost their proteolytic activities, consistent with recent
reports (4, 17, 23, 25). We found that the presence of extra amino
acids at the N terminus did not appear to affect the proteolytic activity of streptopain. Compared with the wild-type enzyme, the activities of mutants Q186N, N356D, and W357A of SPE B decreased by
1.8-, 359-, and 50,000-fold, respectively (Table I). The inactive C192S
mutant was used as a substrate to examine the proteolytic activities of
the native and recombinant streptopain proteins (17). Fig.
4 shows the results of SDS-PAGE analyses
of the processing of the pro-SPE B C192S mutant using the native,
wild-type, Q186N, N356D, and W357A proteins (Fig. 4). Our results show
that recombinant streptopain was as active as the native form (Fig. 4,
A and B). Interestingly, native SPE B was less
efficient at processing the 42-kDa C192S mutant than recombinant SPE B. Based on the N-terminal sequence analyses, recombinant and native SPE B
contain a mixture of products 8 and 9. Different 28-kDa products may
have effects on their trans-processing activities.
The times required for the conversion from the 42-kDa C192S mutant to
the 28-kDa form with recombinant streptopain and the Q186N, N356D,
W357A mutants were 1, 2.5, 14, and 24 h, respectively. Compared
with the wild-type enzyme, the proteolytic activities of the Q186N,
N356D, and W357A mutants decreased by 3-, 10-, and 30-fold,
respectively. The mutations of Gln186, Asn356,
and Trp357 had effects consistent with the results from the
azocasein assay, giving the relative proteolytic activities of the
mutant proteins on the order of Q186N > N356D > W357A > C192S, H340R. As shown in Fig. 4, the trans-processing of
the pro-SPE B C192S mutant by the wild-type and Q186N and N356D
mutant proteins generated at least eight intermediates. In contrast,
the trans-processing of the pro-SPE B C192S mutant by the
W357A mutant gave rise to only two visible intermediates. This finding
indicates that Trp357 plays an important role in substrate
recognition. This result is consistent with the reported x-ray
structure of pro-SPE B, which shows that Trp357,
Phe342, and Phe367 form a hydrophobic
substrate-binding site (26).
Processing of the Pro-SPE B C192S Mutant by 28-kDa
Active Streptopain--
The maturation of pro-SPE B goes through
multiple-step processing involving six intermediates and one final
product (17). Similar intermediates are involved in the
trans-processing of the pro-SPE B C192S mutant by the 28-kDa
active form of streptopain (17). Fig. 4A shows that the
processing of the C192S mutant by streptopain involved at least nine
cleavages. The cleavage sites of these products were determined by an
N-terminal sequencer and are summarized in Table
II. Product 9 was identified as the 28-kDa active form of streptopain, consistent with previous reports (8,
17). However, although previous studies reported the presence of six
intermediates, four of which have been characterized, we identified a
total of eight intermediates. We also found that, based on the
sequences of all intermediates and the final product, the
substrate specificity of streptopain was similar to the substrate preference of the papain-like family, with a preference for a hydrophobic residue (isoleucine (5/9), tyrosine (2/9), methionine (1/9), or valine (1/9)) at the P2 site. Analysis of the cleavage sites
also revealed trends, with an asparagine residue (3/9) at the P3 site,
a lysine residue (3/9) at the P1 site, a glycine residue (3/9) at the
P2' site, a glycine or alanine residue (2/9) at the P3' site, and a
glutamate residue (3/9) at the P4' site (Table II). This pattern of
intermediate accumulations and disappearances indicates that products 8 and 9 of streptopain were the major final products from the processing
of the pro-SPE B C192S mutant by streptopain. The primary sequence of
pro-SPE B and the resulting cleavage sites are shown in Fig.
5.
Processing of the Pro-SPE B C192S Mutant by Plasmin and
Trypsin--
The inactive C192S mutant of pro-SPE B was also used to
examine its digestion process with plasmin and trypsin. To compare the
processing of this mutant with that of the 28-kDa active form of
streptopain, we used the same reaction substrate/enzyme ratio of 20:1.
Fig. 6 shows that trypsin converted the
42-kDa C192S mutant into the 28-kDa form, which contained two products,
and that plasmin converted the 42-kDa C192S mutant into the 28-kDa form, which contained only one product. Both processes involved the same two intermediates; however, the final product differed from
the 28-kDa active mature streptopain, and they were 5 residues apart.
The accumulation of fewer intermediates was in contrast with the
results observed for the processing by streptopain.
The trypsin and plasmin cleavage sites in the primary sequence of
pro-SPE B are shown in Fig. 5, and the N-terminal sequences of the
reaction products with plasmin are summarized in Table II. Trypsin and
plasmin are known to prefer lysine or arginine at the P1 site. The
sequences of the intermediates and the final product are
consistent with their specificity. In contrast, intermediates 1 and 7 (with lysine at the P1 site) observed in streptopain were not
found. The times required for the complete digestion of the 42-kDa
C192S mutant by streptopain, trypsin, and plasmin were 1, 12, and
72 h, respectively. Therefore, the digestion rates of the 42-kDa
C192S mutant by the 28-kDa active form of streptopain, trypsin, and
plasmin were 2, 0.2, and 0.033 nmol/h, respectively. These findings
indicate that the 28-kDa active form of streptopain likely plays the
most important role in activating pro-SPE B under physiological conditions.
Autocatalysis of Pro-SPE B Mutants--
To study the effect of
mutation on the autocatalysis of pro-SPE B, we analyzed the conversion
of the 42-kDa mutant protein to the 28-kDa active form. As shown in
Fig. 7, the C192S and H340R mutants,
which had no proteolytic activities, also had no autoprocessing activities. The Q186N mutant was converted into the 28-kDa form within
1 h, and the W357A mutant was converted within 14 h. Note that the conversion rate of the W357A mutant was 10-15-fold slower than that of the Q186N mutant. In contrast, the 42-kDa N356D mutant showed no autoprocessing even after 14 h, and the protease
activity of the N356D mutant was higher than that of the W357A mutant
(Table I). These results indicate that Asn356 of pro-SPE B
is involved in the cis-processing of the propeptide. Based
on the reported x-ray structure of the C192S mutant, Asn356
does not have direct contact with the propeptide region because the
Asn356 side chain does not face the propeptide region (26).
Interestingly, the corresponding residue of papain is not
essential for activity and likely plays a role in positioning the
catalytic residues (18). This study shows a new functional role for
this conserved asparagine residue of the cysteine protease family.
cis- and trans-Processing of Pro-SPE B--
As described above,
the autocatalysis of pro-SPE B is either a cis- or
trans-processing mechanism or, more likely, involves a
combination of the two. The cis-processing portion of the
mechanism is likely characterized by a zero-order reaction, where the
precursor-processing rate is independent of the precursor
concentration. In contrast, the trans-processing portion of
the mechanism is likely characterized by a higher order reaction, where
the precursor-processing rate is dependent on the precursor
concentration. In this study, we used pro-SPE B to measure the initial
rate of precursor processing at concentrations of 0.2, 0.35, 0.5, 0.75, 1, 2, 3, and 4 µM. The initial rate of precursor
processing was determined by measuring the depletion of the precursor
over time, and changes in the precursor bands were quantified using an
imaging system. We found that the rate of transformation from the
42-kDa zymogen to the 28-kDa enzyme was faster at higher
concentrations, indicating trans-processing. Fig.
8 shows the plot of the relative rate of
activation versus the concentration of precursor as fitted
by linear regression. This plot demonstrates that the reaction
mechanism involves cis-processing because the extrapolated
rate was not null at the zero pro-SPE B concentration. This plot also
demonstrates that the reaction mechanism additionally involves
trans-processing because the rate of activation was pro-SPE
B concentration-dependent. From these data, we can conclude
that the autoactivation of pro-SPE B results from a combination of
cis- and trans-processing.
Fig. 8 shows that the extrapolated rate is 0.7583 when the
concentration of pro-SPE B is zero and the slope is 22.833. Therefore, it can be calculated that the relative rates of cis- and
trans-processing (0.7583) are equal at a pro-SPE B
concentration of 33.2 nM. This concentration is 3.3-fold
lower than with propapain, which is 110.8 nM (20). These
results indicate that the maturation of streptopain is similar to that
of propapain and involves both cis- and
trans-processing.
To understand the role of streptopain in the pathogenesis of GAS,
one must first understand the maturation processing of pro-SPE B to the
28-kDa active form of streptopain. The steps involved in the maturation
of zymogens for the papain-like family of cysteine proteases have been
well documented (18-20, 31-33). On the basis of the consensus
sequence and the sizes of the prodomain, streptopain belongs to the
C10 family of cysteine protease clan CA, and papain belongs
to the C1 family (20, 21, 34, 35). To better understand the
maturation of the streptopain protein, we expressed the wild-type and
mutant proteins of streptopain in E. coli and purified them to homogeneity. Our CD studies showed that the wild-type and mutant proteins of streptopain expressed in E. coli retained their
structural integrity. Recombinant streptopain was expressed as a 42-kDa
zymogen and converted into a 28-kDa enzyme during the course of
purification. 1 mM HgCl2 can be used as
inhibitor during purification to prevent the conversion. Our protease
assays showed that recombinant 28-kDa streptopain was as active as
native streptopain. The presence of extra amino acids at the N terminus
did not appear to affect the protease activity of recombinant
streptopain or the maturation processing of pro-SPE B mutants. CD
analyses showed that the secondary structures of the mutant proteins
are similar to the reported three-dimensional structure of the pro-SPE
B C192S mutant. To our knowledge, these findings provide the first
direct evidence that highly labile streptopain can be expressed in
E. coli and purified to homogeneity while maintaining its
structural integrity and full activity.
Many studies have shown that the maturation of cysteine protease family
members, including propapain and procathepsins B, K, and L, involves
both cis- and trans-processing (20, 31-33). The
effect of the pro-SPE B concentration on the rate of processing and the
proteolytic processing of pro-SPE B mutants as shown in this study
demonstrates that the autoactivation mechanism of pro-SPE B is stepwise
and involves at least eight intermediates with a combination of
cis- and trans-processing. Based on the sequences of these intermediates, the specificity pocket of streptopain is
similar to that of the papain-like family, with preferences for a
hydrophobic residue at the P2 site. In the maturation of pro-SPE B
mutants, only Q186N and W357A were transformed from zymogens into the
28-kDa active forms. In contrast, N356D was not autocatalytically
converted into the 28-kDa active form. However, N356D exhibited
protease activity, which converted the pro-SPE B C192S mutant into the
28-kDa form. Based on the reported x-ray structure of the C192S
mutant, Asn356 does not have direct contact with the
propeptide region because the Asn356 side chain does
not face the propeptide region (26). These results suggest that
Asn356 of pro-SPE B is involved in the
cis-processing of the propeptide.
Although the maturation processing of pro-SPE B by exogenous proteases
has been shown (12, 14, 17), no studies have been performed to compare
the maturation processing of streptopain by both autocatalysis and
exogenous proteases. In this study, we found that both trypsin and
plasmin could convert pro-SPE B into the 28-kDa active form with three
and two intermediates, respectively. Although the maturation processing
of the pro-SPE B C192S mutant by exogenous proteases produced fewer
intermediates, the rates of digestion of the 42-kDa C192S mutant by
trypsin and plasmin were 10- and 60-fold slower than that of the 28-kDa
active form. Analyses of the numbers and sequences of all intermediates and the final products created by streptopain, plasmin, and trypsin revealed that streptopain is a relatively promiscuous protease. We
speculate that 28-kDa active form of streptopain plays the most
important role in converting pro-SPE B into 28-kDa streptopain under
physiological conditions. However, sequence analysis of the SPE B gene
from 200 GAS isolates showed that 20% of these variants contain an
Arg-Gly-Asp motif that preferentially binds integrins
Compared with the maturation of procathepsins, the maturation
processing of pro-SPE B involves many more identifiable intermediates. In contrast, the accumulation of multiple intermediates is not found in
propapain or procathepsins (20, 31-33). The biological significance of
having so many intermediates involved in the autocatalytic processing
of pro-SPE B is still unclear. Because the propeptide region of pro-SPE
B is very close to the substrate-binding site, it is likely that these
intermediates with different prosegment sizes likely have different
substrate specificities. This is consistent with the results from our
proteolytic studies that streptopain has diverse substrate specificity.
The relative rates of the cis- and
trans-processing of procathepsin L, propapain, and pro-SPE B
are equal at concentrations of 0.24, 110.8, and 33.2 nM,
respectively (20, 32). These results indicate that the maturation of
pro-SPE B, propapain, and procathepsin L involves both cis-
and trans-processing. Regulation of the cysteine protease
activity of the papain-like family has been implicated in a wide
variety of human diseases (35, 36). Specifically, rapidly accumulating
evidence suggests that the growth and proliferation of pathogenic
bacteria depend on proteolytic enzymes of the invading organism (2, 33,
37, 38). Because the 28-kDa active form of streptopain plays an important role in GAS pathogenesis (12, 14, 17), continuing studies
regarding the maturation of pro-SPE B are increasingly important.
In conclusion, we expressed wild-type and Q186N, C192S, H340R, N356D,
and W357A mutant streptopain in E. coli and purified them to
homogeneity. We found that streptopain produced in E. coli
possesses the same function and structure as the native protein. This
is the first report to show that labile streptopain can be expressed in
E. coli with the correct fold. We also found that the
maturation of pro-SPE B is stepwise and involves at least eight
intermediates that involve a combination of cis- and
trans-processing. Like other papain-like family members of
cysteine proteases, including propapain and procathepsin L, the
maturation processing of pro-SPE B involves a combination of
cis- and trans-processing. Compared with other
exogenous proteases, the 28-kDa active form of streptopain is the most
effective protease for processing pro-SPE B. We speculate that a
similar mechanism occurs in vivo. Because streptopain is a
possible drug target in the control of streptococcal infection, this
study serves as the basis for gaining insight into streptococcal infections by exploring the structure-function relationships of streptopain. This study also extends our understanding of the molecular
basis of the maturation mechanism of streptopain under physiological conditions.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Ribbon drawing of the three-dimensional
structure of pro-SPE B. The -helices,
-strands, and
loop regions of streptopain are shown in red,
blue, and gray, respectively. The residues of the
P1 (Asn68, Tyr79, Val80,
Ala118, and Lys129) and P2 (Val67,
Met78, Ile117, and Ile128) cleavage
sites of the proregion observed in the maturation processing of pro-SPE
B are shown in dark blue. The mutated
Gln186, Cys192, His340,
Asn356, and Trp357 residues of streptopain are
shown in green. The structure figure was prepared using the
program MOLMOL (39).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (1 mM), and the culture was further incubated at 15-37 °C
for 2-24 h to induce protein production. Cells were harvested by
centrifugation and lysed by liquid shear with a French press to obtain
the extract.
20 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C for >2 years. The experimental molecular mass of the
C192S mutant was determined to 42,334 Da with a deviation of
<0.5 compared with the calculated value.
Expression and purification of wild-type streptopain and its Q186N,
C192S, H340R, N356D, and W357A mutants
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Fig. 2.
SDS-PAGE analysis of the wild-type and C192S,
H340R, N356D, Q186N, and W357A mutant proteins of streptopain.
Lane M, molecular mass markers (94, 67, 43, 30, and 20.1 kDa); lane 1, wild-type streptopain; lane 2,
C192S mutant; lane 3, H340R mutant; lane 4, N356D
mutant; lane 5, Q186N mutant; lane 6, W357A
mutant. The proteins loaded in each lane were normalized.
-helix, 55.5 and 52.5%
-structure, and 25.7 and 27.3% coil,
respectively (Fig. 3A). These
values are consistent with the reported x-ray structure of the pro-SPE
B C192S mutant, which can be calculated by ignoring the propeptide
region (26). In addition, the CD spectra of the 42-kDa zymogen forms of
Q186N, C192S, H340R, N356D, and W357A were similar (Fig.
3B). CD analysis showed that they contained 19.1-23.5%
-helix, 51.5-54.2%
-structure, and 22.2-25.2% coil, respectively. Their secondary structures obtained from CD spectra were
similar to the reported three-dimensional structure of the pro-SPE B
C192S mutant, which contains 23.6%
-helix, 52.5%
-structure, and 23.9% coil (26).
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Fig. 3.
CD analyses of the native and recombinant
wild-type 28-kDa forms (A) and the 42-kDa forms of the
Q186N, C192S, H340R, N356D, and W357A mutant proteins
(B) of streptopain. All samples contained 1 mg/ml
protein in PBS at 27 °C. rSPE B, recombinant SPE B.
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Fig. 4.
SDS-PAGE analysis of the digestion of the
pro-SPE B C192S mutant by native (A) and recombinant
wild-type (B) streptopain and the Q186N
(C), N356D (D), and W357A
(E) mutants. The native or recombinant
(r) streptopain protein at a final concentration of 1.2 µM was incubated with the pro-SPE B C192S mutant (24 µM) for various time intervals. Lane M,
molecular mass markers (43 and 30 kDa); lanes 1-5
and 6, the pro-SPE B C192S mutant incubated at 37 °C in
the presence and absence of active enzymes, respectively; lane
7, active enzyme. The cleavage products of the reaction are
indicated.
Sites of cleavage of the proSPE B C192S mutant by streptopain and
plasmin
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Fig. 5.
Primary sequence of recombinant streptopain
and the sites of cleavage of pro-SPE B by mature streptopain, plasmin,
and trypsin (A) and schematic representation of
pro-SPE B (B). The sequence of recombinant
streptopain is shown and contains 11 extra vector residues, 6 histidine
residues, and sequences without signal peptide. For purposes of
comparison with previous mutagenesis studies, in this study, we
numbered the first methionine of the signal peptide of streptopain as
position 1. The boxed sequence represents the proregion. The
thin, double-tailed, thick, and
gray arrows designate the sites of cleavage by streptopain;
by streptopain, trypsin, and plasmin; by streptopain and trypsin; and
by trypsin and plasmin, respectively.
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Fig. 6.
SDS-PAGE analysis of the digestion of the
pro-SPE B C192S mutant by trypsin (A) and plasmin
(B). A final concentration of 1.2 µM trypsin or plasmin was incubated with the pro-SPE B
C192S mutant (24 µM) for various time intervals.
Lane M, molecular mass markers (94, 67, 43, 30, and 20.1 kDa); lanes 1-6 (A) and 1-7
(B), the pro-SPE B C192S mutant incubated at 37 °C in the
presence of trypsin or plasmin, respectively; lane 7 (A), the pro-SPE B C192S mutant incubated at 37 °C in the
absence of proteases.
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Fig. 7.
SDS-PAGE analysis of the autoprocessing of
the C192S, H340R, N356D, Q186N, and W357A mutants of pro-SPE B. Lane 1, purified wild-type streptopain; lanes
2-4, the C192S, H340R, and N356D mutants of pro-SPE B incubated
for 24 h; lane 5, the Q186N mutant of pro-SPE B
incubated for 1 h; lane 6, the W357A mutant of pro-SPE
B incubated for 14 h. All samples contained 16 µM
protein in PBS with 5 mM EDTA and 5 mM DTT
incubated at 37 °C.
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Fig. 8.
A, plot of the initial rate of pro-SPE B
processing versus precursor concentrations. The calculated
data points were fitted by linear regression. The S.D. values of the
relative rates of activation are shown. B, expanded plot for
the pro-SPE B concentrations of 0.2, 0.35, and 0.5 µM.
The dotted line shows that the relative rates of
cis- and trans-processing are equal at a pro-SPE
B concentration of 33.2 nM.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3 and
IIb
3
(28). Angiostatin, a plasmin fragment containing three to four
N-terminal kringle domains, is a ligand for integrin
v
3 (40). Therefore, integrin binding may
concentrate both streptopain and plasmin at the human cell surface,
thereby accelerating the maturation of streptopain. These interactions may be important because they may provide a means for GAS invasion.
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FOOTNOTES |
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* This work was supported by National Health Research Institutes Grant NHRI-GT-EX90-9027SP and National Cheng Kung University Hospital Grants 89-04 and 90-04.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 886-6-235-3535 (ext. 5515); Fax: 886-6-274-1694; E-mail: wjcnmr@mail.ncku. edu.tw.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M209038200
2 Available at merops.sanger.ac.uk/.
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ABBREVIATIONS |
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The abbreviations used are: GAS, group A streptococcus; SPE B, streptococcal pyrogenic exotoxin B; PBS, phosphate-buffered saline; DTT, dithiothreitol.
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