Maturation Processing and Characterization of Streptopain*

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. In-terestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn 356 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

Group A streptococcus (GAS) 1 causes several human diseases, including as pharyngitis, acute rheumatic fever, scarlet fever, post-streptococcal glomerulonephritis, and toxic shocklike 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)(4)(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)(13)(14)(15)(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)(18)(19)(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 (C 10 ) 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, Ͼ60 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 C 10 and C 1 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 (Cys 192 , His 340 , and Asn 356 ) occur in the same order as those in papain (Cys 25 , His 159 , and Asn 175 ), 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)(24)(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: Gln 186 is the residue outside the substrate-binding pocket and contacts with the loop containing Cys 192 ; Cys 192 and His 340 are the catalytic residues; and Asn 356 and Trp 357 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 Cys 192 or His 340 of streptopain leads to a complete loss of protease activity (4,23,25). Asn 175 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 Trp 357 may replace the function of Asn 175 in papain (26). The crystal structure also demonstrates that Phe 342 , Trp 357 , and Phe 367 form a hydrophobic pocket that fits a hydrophobic residue at the P2 site of the substrate (26). Although the role of Asn 356 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.

EXPERIMENTAL PROCEDURES
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 5Ј-G-GATCCGGATCCCATCATCATCATCATCATGATCAAAACTTTGCTC-GTAACGAA-3Ј with a His 6 tag and BamHI recognition and by the antisense primer 5Ј-GGATCCGGATCCCTAAGGTTTGATGCCTACAA-CAG-3Ј 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 A 600 ϭ 0.5-1.0. To the culture was added isopropyl-1-thio-␤-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.
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 ϫ 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 A 280 Ͻ 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 Ni 2ϩ 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 Ϫ20°C.
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-m 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 l of streptopain or mutant protein to 160 l 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 l 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 g of soluble azopeptide/min. The specific absorption coefficient (A 366 1% ϭ 40) of the azocasein solution was calculated by measuring its absorption after total digestion (28).
Processing 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 l of PBS containing 5 mM EDTA and 5 mM DTT. Purified or recombinant streptopain protein at a final concentration of 1.2 M was incubated with the 42-kDa C192S mutant (24 M) at 37°C for the designated time intervals ranging from 0 to 14 h. The reactions were quenched by addition of 5 l of 50 M E-64 (transepoxysuccinyl-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 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 leastsquare fitting program (LINCOMB) as described by Perczel et al. (29,30).

RESULTS
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 Ni 2ϩ 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 HgCl 2 was added as an inhibitor to prevent the conversion. In contrast, all mutant proteins were purified as 42-kDa zymogens without adding HgCl 2 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 rela-tive 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 ϳ45, 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 Ϫ20°C for Ͼ2 years. The experimental molecular mass of the C192S mutant was determined to 42,334 Da with a deviation of Ͻ0.5 compared with the calculated value.
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 (LIN-COMB) of Perczel et al. (29,30). Although a slight difference at ϳ210 nm was observed, CD analysis showed that recombinant and native 28-kDa streptopain had 18.8 and 20.2% ␣-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).
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 Gln 186 , Asn 356 , and Trp 357 had effects consistent with the results from the azocasein assay, giving the relative proteolytic activities of the mutant proteins on the order of Q186N Ͼ N356D Ͼ W357A Ͼ 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 Trp 357 plays an important role in substrate recognition. This result is consistent with the reported x-ray structure of pro-SPE B, which shows that Trp 357 , Phe 342 , and Phe 367 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 P2Ј site, a glycine or alanine residue (2/9) at the P3Ј site, and a glutamate residue (3/9) at the P4Ј 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.
2/9 3/9 5/9 3/9 3/9 2/9 3/9 By plasmin These cleavage sites were reported by Doran et al. (17). b These intermediates were identified in this study. c C represents the preferred amino acids at different sites. d X represents any amino acid. e F represents the fractions of the preferred amino acids at different sites.

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.
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 Asn 356 of pro-SPE B is involved in the cis-processing of the propeptide. Based on the reported x-ray structure of the C192S mutant, Asn 356 does not have direct contact with the propeptide region because the Asn 356 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 precursorprocessing 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 M. 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 cisprocessing 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. DISCUSSION 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 C 10 family of cysteine protease clan CA, and papain belongs to the C 1 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 HgCl 2 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)(32)(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, Asn 356 does not have direct contact with the propeptide region because the Asn 356 side chain does not face the propeptide region (26). These results suggest that Asn 356 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 ␣ 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.
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)(32)(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 transprocessing. 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 strep-topain 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.