Abstract
V8 protease (GluV8), a member of the glutamyl endopeptidase I family, isolated from the V8 strain of Staphylococcus aureus, is widely utilized for proteome analysis because of its unique substrate specificity and resistance to detergents. We recently developed an Escherichia coli expression system for the production of GluV8 based on a technique that suppresses the auto-proteolysis: the use of the prosequence of its homolog (GluSE) from Staphylococcus epidermidis as a chimeric form or the introduction of 4 substitutions in the prosequence of GluV8. In the present study, we refined this
Introduction
Staphylococcus aureus produces extracellular proteases, which are regarded as important virulence factors. One of the classically defined endoproteases is a serine protease, GluV8 1 , known also as V8
protease/Glu-C/SspA [1] . An S. aureus GluV8-deficient mutant was severely attenuated in virulence in mouse abscess, bacteremia, and wound infection models [2] . S. aureus proteases modulate bacterial adhesive molecules, changing the phenotype from adhesive to invasive, and degrade fibronectin-binding proteins and protein A [2] , which are adhesion molecules on the staphylococcal surface [3] .
GluV8 belongs to the glutamyl endopeptidase I (EC 3.4.21. 19 ) family and specifically cleaves the peptide bond after negatively charged residues, glutamic acid, and less potently, aspartic acid [4] . The nucleotide sequence encodes a protein of 336 amino acids that includes a prepropeptide consisting of 68 residues (Met 1 -Asn 68 ) and a C-terminal tail of 52 residues containing a 12-fold repeat of the tripeptide Pro-Asp/Asn-Asn [5] . Drapeau [6] reported that activation of the GluV8 precursor is achieved by a neutral metalloprotease. Shaw et al. [7] further demonstrated that the cascade reactions in processing of major extracellular pathogenic proteases of S. aureus from metalloprotease/aureolysin, GluV8/SspA, and finally, to cysteine protease/SspB.
We previously purified [8] and cloned [9] the glutamyl endopeptidase from Staphylococcus epidermidis (designated GluSE), which consists of 282 amino acids composed of a preprosequence (Met 1 -Ser 66 ) and mature portion (Val 67 -Gln 282 ). GluSE does not have the C-terminal repeat sequence found in GluV8 [9] . Another Staphylococcal homolog, glutamyl endopeptidase from Staphylococcus warneri (designated GluSW), was cloned and deduced to be composed of 316 amino acids and to possess a 34-amino acid Asp/Asn-rich region at its C-terminus [10] . When GluV8 with its C-terminal repeat deleted was expressed in E. coli, it could fold to an active form after denaturation-renaturation 5 treatment [11] . Thus, the C-terminal repeat sequence is not needed for the enzymatic activity and its role remains unknown. The presequence of GluV8 (Met 1 -Ala 29 ) functions as a signal peptide required for the secretion, and the prosequence (Leu 30 -Asn 68 ) for the suppression of the protease activity [12, 13] .
A recent study proposed the sequential processing within the prosequence of GluV8 [12] .
Expression of recombinant GluV8 in Escherichia coli would be useful to elucidate in detail the roles of the prepro-and C-terminal repeated segments as well as the specific amino acid residues involved in the processing and enzymatic activity. For instance, we have recently demonstrated the importance of the N-terminal Val 69 and Ser 237 by in vitro mutagenesis studies [13] . Because secreted bacterial proteases including GluV8 are neither membrane-bound nor glycosylated, an E. coli expression system should be suitable for their expression.
There are 2 major problems that could occur on the E. coli expression of proteases. At first, the expression of a target protein occasionally produces insoluble materials called inclusion bodies. Prior to our recent studies [13, 14] , one E. coli expression study was reported [11] , in which mature GluV8 was expressed as a fusion protein tagged to exogenous peptides at N-and C-termini for prevention of its auto-activation. This form was expressed and purified as inclusion bodies. The mature protein was then obtained by the cleavage of the exogenous peptides, denaturation-renaturation, and then ion-exchange chromatography for purification. Because this procedure is time-consuming and the recovery at the denaturation-renaturation step is low (20%), this is not generally employed for the purification. The commercially available GluV8 is still purified from the culture supernatant of the S. aureus V8 strain.
As a second problem, even if a target protein is expressed as a soluble form, the protease might be expressed or converted to a mature form in host cells. In particular, if the maturation occurs through 6 an auto-proteolytic pathway, the expression of a target protease may be toxic for host cells.
Accordingly, one of the ideal expression systems should be the expression of a soluble and latent form of a protease in E. coli and the subsequent in vitro maturation processing.
We recently developed an expression system for GluSE in E. coli [13, 14] . GluSE was expressed as a full-length form with a His hexamer tag at its C-terminus. The purified GluSE was recovered as enzymatically inactive proforms, which contained the full or parts of its prosegment. In contrast, GluV8 was poorly expressed by the identical system due to auto-degradation. The comparison of their prosequences suggested auto-proteolytic degradations of the GluV8 prosequence at the Glu 62 -Lys 63 and
Glu 65 -Arg 66 bonds (Fig. 1a) . In fact, GluV8 was efficiently recovered by swapping its preprosegment with that of GluSE or by the introduction of 4 amino acid substitutions to the prosequence of GluV8 [13] . However, the expression level of GluV8 was still lower than that of GluSE. Considering the low similarity of the prosequences between GluSE and GluV8, we speculated that the prepropeptide of GluSE can not completely compensate the role of that of GluV8 and that the 4 substitutions did not fully suppress the auto-degradation.
In the present study, to evaluate the auto-degradation occurring during the expression, we utilized a constitutively inactive form of GluV8, the active Ser 237 of which was substituted to Ala [13] . By use of this form as a positive control, we established an optimal expression system that completely suppressed the auto-degradation of GluV8 in E. coli. We also tried to express another member of the family proteases, GluSW, which possessed 3 Glu and 2 Asp within the prosequence. The identification of the N-terminus of its mature form would be helpful for the understanding of the functioning mechanism of GluSW.
Materials and methods

Materials
The materials used and their sources were the following: Expression vector pQE60 and plasmid 
Bacterial expression vectors
All recombinant proteins were expressed in E. coli with a His hexamer tag at their C-terminus by use of the pQE60 expression vector. The constructs and their modified positions are summarized (Table 1) .
Genomic DNA was purified from S. warneri JCM 2415 as described previously [15] . The DNA fragment encoding the full-length form of GluSW was amplified by PCR with a pair of primers (sense 5'-TTTGGATCCAAAGTTAAGTTTTTTACAGCA and anti-sense, 5'-GGCGGATCCCGCTGCGTCTGGATTATCGTA). BamHI sites are indicated by italic letters. The resultant PCR fragment was inserted into a BamHI site of pQE60 after being cleaved with BamHI.
Y1090
[pREP4] cells were transformed with the plasmid. Expressions of GluSE, GluV8, a chimeric 8 protein of prepro-GluSE and mature GluV8, GluV8 with 2 (GluV8mut2) or 4 (GluV8mut4) amino acid substitutions, and GluSE and GluV8 with the substitution at their active Ser were described previously [13, 14] .
The plasmids encoding GluV8 with the 5 amino acid substitutions (designated GluV8mut5) and
GluV8mut5 with its C-terminal repeat deleted (designated GluV8mut5ΔC) were prepared by PCR with pQE60-GluV8mut4 and GluV8mut4ΔC as templates with sense 
Expression and purification of recombinant proteins
Recombinant proteins were expressed and purified as described previously [16] . Briefly, E. coli Y1090[pREP4] (300 ml) carrying pQE60-derived expression plasmids was cultured overnight at 37˚C
in LB broth containing 50 μg/ml of ampicillin and 25 μg/ml of kanamycin. Protein expression was induced by dilution of the culture with LB broth (500 ml) containing 0.2 mM isopropyl β-D-thiogalactopyranoside and 50 μg/ml of ampicillin and incubation at 30˚C for 3 h. Fig. 2 ), they were expressed as described above in the following volumes: An overnight culture (3 ml) of cells was diluted to 10 ml for induction. After harvest, the cells were lysed with 1 ml of the lysis solution and the lysate (1 ml) was adsorbed onto a Talon affinity resin (20 µl of bed volume) for 5 min. After extensive washing of the unbound materials, 40 µl of SDS-buffer was added. The resin was then heated to denature bound proteins. Thereafter, aliquots (5 μl) of the lysates or bound proteins were separated by SDS-PAGE.
The intensity of CBB-stained bands of purified proteins was quantified by use of PDQuest 2-D Analysis
Software (Bio-Rad).
In vitro processing and the measurement of protease activity
Unless otherwise stated, the in vitro processing of recombinant proteins and subsequent protease assay were performed as follows: Recombinant proteins (3 μM) were incubated in 0.1 ml of 10 mM sodium borate, pH 8.0, 0.005% (v/v) Triton X100 containing 2 mM CaCl 2 with 2.8 (ratio 110:1) or 8.4
pmol (ratio 37:1) of thermolysin at 37˚C for 4 h. Thereafter, aliquots of glutamic acid-specific proteases (40 nM) were incubated with 20 μM Z-Leu-Leu-Glu-MCA in 0.2 ml of 50 mM Tris-HCl (pH 8.0) and 5 mM EDTA at 25˚C for 2 h. The fluorescence was measured with an excitation at 380 nm and emission at 460 nm with a Fluorescence Photometer F-4000 (Hitachi, Tokyo, Japan).
Protease activity was also determined by colorimetric measurement by use of azocasein (Sigma).
Following the incubation of the proteases (57 pmol/75 μl) with 2 mg/ml of azocasein in 50 mM
Tris-HCl (pH 8.0) containing 5 mM EDTA at 37˚C for 1 h, the reaction was stopped by the addition of 15% (w/v) ice-cold trichloroacetic acid at a final concentration of 4% (w/v). After a 10-min incubation at 0˚C, the supernatant obtained by centrifugation at 10,000 x g for 5 min was mixed with an equal volume of 0.5 M NaOH, and then the absorbance at 440 nm was measured.
Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)
Purified proteins (0.8 μg) were separated by PAGE in the presence of 0.1% SDS at a polyacrylamide concentration of 12% (w/v). In the experiments of Figs. 2 and 5, aliquots (5 μl) of the lysates or purified fractions were loaded onto an SDS-PAGE gel. Separated proteins were stained with Coomassie Brilliant Blue (CBB) or subjected to the immunoblotting with anti-penta His mAb (0.2 μg/ml) as described previously [13] .
N-terminal amino acid sequencing
N-terminal amino acid sequences were determined after separation of recombinant proteins by SDS-PAGE and transference to a polyvinylidene difluoride membrane (Sequi-Blot PVDF Membrane, Bio-Rad). After having been stained with CBB, the bands were excised and directly sequenced with a model Precise 49XcLC protein sequencer (ABI).
Protein Concentration
Protein concentrations were determined by the bicinchoninic acid method (Pierce). Bovine serum albumin dissolved in 0.1 M imidazole (pH 8) and 10% (v/v) glycerol was used as a reference to adjust buffer compositions to affinity-purified samples.
Results

Comparison of the expression levels among the glutamyl endopeptidases in E. coli
Comparison of the amino acid sequences of the GluSE, GluV8, and GluSW except for the C-terminally extra regions revealed the highest homology at the mature sequence (58.8 to 80.1%),
followed by the presequence (29.6 to 55.2%), and lowest at the prosequence (15.4 to 51.3%) (Fig. 1) .
Among them, the mature sequences are the most conserved between GluV8 and GluSW (80.1%),
indicating their close kinship. The homology of the prosequence is also highest between GluV8 and GluSW (51.3%).
GluSE, GluV8, GluSW, and their derivatives (Table 1) were expressed in E. coli with a C-terminal
His hexamer tag. The immunoblotting analysis of the lysates revealed that all of the recombinant proteins had been produced (Fig. 2a) . The appearance of several bands in most lanes indicated their partial degradation. CBB-staining of bacterial lysate proteins revealed 32-kDa (Fig. 2a, lanes 1, 7, 8 , and 10) and 38-kDa (lane 12) bands distinguished from those of endogenous bacterial proteins.
In order to quantify the expression, we performed batch purification with 1ml of the lysate (Fig.   2b ). The intensity of the purified bands may be considered to reflect their expression levels. GluSE was expressed efficiently (Fig. 2b, lane 1) , whereas GluV8 was scarcely expressed (lane 2). When the chimeric protein GluSE-V8, i.e., the prepro-and mature sequences of which were derived from GluSE and GluV8, respectively, was expressed, the density of the 44-kDa band was increased, indicating the suppression of degradation (lane 3).
Enhancement of the expression of recombinant GluV8 by modification of the prosegment
Instead of using the prepropeptide of GluSE, we serially substituted in the GluV8 prepropeptide 13 several amino acids constituting the peptide bonds potentially auto-proteolysed to those located at the equivalent positions in GluSE (Fig. 1a) . At first, 2 amino acid substitutions, i.e., from Glu 62 and Glu 65
to Gln 62 and Ser 65 (GluV8mut2) partially increased the density of the 39-44 kDa bands (Fig. 2b, lane 4) compared with the density of the bands of GluV8 possessing its authentic prepropeptide (lane 2).
When 2 additional amino acids, i.e., Ala 67 and Asn 68 , were substituted to Pro 67 and Ser 68 (GluV8mut4), the 44-kDa band density increased (Fig. 2b, lane 5) . We further expressed GluV8mut5, which carried an additional substitution of Asp 36 to His. GluV8mut5 (Fig. 2b, lane 6) showed a 44-kDa band having much higher density than that of GluV8mut4 (lane 5). Concomitantly, the degradation products smaller than 44 kDa of GluV8 and GluV8mut2 (Fig. 2a , lower panel, lanes 2 and 4) disappeared on GluV8mut4 and GluV8mut5 (lanes 5 and 6). We postulated that the deletion of the C-terminal repeat sequence might further enhance the expression level of GluV8, because a shorter polypeptide might be more efficiently translated in E. coli. In fact, the expression level of GluV8mut5ΔC was additionally increased (Fig. 2b, lane 7) .
Expression of the constitutively inactive form of GluSE and GluV8
It remained unknown whether the auto-proteolysis was completely suppressed by the 5 substitutions or further suppression was possible. To settle this issue, we compared the expression level of that molecule to that of the constitutively inactive form, the active Ser residue of which was substituted to Ala, because the expression level of the inactive form would be expected to be maximal.
In fact, the constitutively inactive forms of GluSE and GluV8 were abundantly expressed in E. coli (Fig.   2, lanes 8-10) . The band densities of GluV8mut5 and GluV8mut5ΔC (lanes 6 and 7) were comparable to those of GluV8mut4 Ser237Ala and GluV8mut4ΔC Ser237Ala (lanes 9 and 10), respectively.
Taken together, these results indicate that the auto-degradation was completely suppressed in GluV8mut5 and GluV8mut5ΔC.
Expression of GluSW
The E. coli expression system of GluV8 developed herein may be applicable to the expression of other members. As an initial attempt, we applied this technique to the glutamyl endopeptidase from S.
warneri, GluSW. The full-length form of GluSW was recovered as 35-, 38-and 40-kDa species (Fig.   2b , lane 11), indicating proteolytic degradation. Because there are Glu residues at positions 28, 60, and 63 and Asp ones at positions 34 and 51, the substitutions of the 5 amino acids to non-auto-cleavable ones were postulated to enhance its expression level. However, we here employed another strategy:
The amino acid sequence at the GluSW proregion is 51.3% identical to the corresponding region of GluV8. Therefore, we suspected that the prosequence of GluV8mut5 might compensate this role. In fact, a chimeric protein carrying the mutated preprosequence of GluV8 and mature sequence of GluSW (designated as GluV8mut5-SW) was efficiently expressed in E. coli as a 38-kDa species (Fig. 2, lane   12 ).
Large-scale expression of GluV8mut5ΔC and GluV8mut5-SW in E. coli
When the wild-type GluV8 and GluV8mut5ΔC were expressed in 800-ml cultures, the expression of GluV8mut5ΔC was much higher than that of GluV8 (Fig. 3a) . Similarly, the expression of GluV8mut5-SW was much higher than that of the wild-type GluSW (Fig. 3b) . Approximately, 20 fg of recombinant GluV8mut5ΔC and 30 fg of GluV8mut5-SW were purified per an E. coli
In vitro processing of the proteases by thermolysin
It has been reported that GluV8 is processed by aureolysin, a thermolysin-family metalloprotease [6, 7] . This processing was faithfully reproduced by thermolysin treatment of the recombinant proteins in vitro (Fig. 4a) . The proteolytic activity toward Z-Leu-Leu-Glu-MCA was highest for GluV8 (Fig.   4b ). The activity of GluSW was slightly higher than that of GluSE (Fig. 4c ). The extremely high activity of GluV8, however, was dependent on substrates, because the proteolytic activity among the proteases toward azocasein showed only a 1.7-fold difference in range (Fig. 4d ). Nevertheless, it
should be noted that the order of the protease activity of the 3 proteases toward azocasein was consistent with their order toward Z-Leu-Leu-Glu-MCA.
Identification of the N-terminus of mature GluSW
Various polypeptides maintaining truncated prosequences were transiently produced during the processing of pro-glutamyl endopeptidases and finally, mature forms with the N-termini of Val 67 for GluSE [14] , Val 69 for GluV8 [12, 13] , and their equivalent amino acid, Leu 48 , for the protease from
Enterococcus faecalris [17] were produced. In contrast, the 31-kDa mature form of GluSW was reported to retain Arg 64 -Ala-Asn 66 at its N-terminus [10] , which is equivalent to the C-terminal tripeptide, His 66 -Ala-Asn 68 , of the GluV8 prosequence (Fig. 1a) . If this form were truly the mature enzyme, GluSW may be activated by the auto-proteolytic cleavage at the Glu 63 -Arg 64 bond (Fig. 1a, an arrow with X). Nevertheless, referred to our recent findings on recombinant GluV8 and GluSE [13, 14] , it was speculated that the Asn 66 -Val 67 bond is the true maturation processing site for GluSW. To settle these alternatives, we determined the proteolytic activity and the N-terminal sequence of recombinant GluSW before and after thermolysin treatment. The 35-kDa wild type GluSW and 38-kDa GluV8mut5-SW possessed no proteolytic activity and the 33-kDa form produced by thermolysin treatment exerted the proteolytic activity (Fig. 5) . The N-terminal sequence of the 35-kDa wild-type GluSW was Arg 64 -Ala-Asn-Val-Ile-Leu-Pro-Asn-Asn 72 , indicating auto-degradation at the Glu 63 -Arg 64 bond. In contrast, the N-terminal sequence of the 38-kDa GluV8mut5-SW was Leu 30 -Ser-Ser-Lys-Ala-Met-His-Gln 37 , demonstrating the proper processing at the pre-pro border (Fig.   1a ). The N-terminal sequence of the enzymatically-active 33-kDa form of wild-type GluSW started from Val 67 . Taken together, these data indicate that the N-terminus of mature GluSW was not Arg 64 , but Val 67 .
Mechanism of the amino acid substitutions responsible for the enhanced expression of GluV8
In addition to the conversion of acidic amino acids to other ones, the replacement of Ala 67 -Asn 68 to
Pro-Ser contributed to the stabilization of GluV8 (Fig. 2, lanes 3 and 4) . Why did this replacement enhance the recovery? We hypothesized that the processing site, Asn 68 -Val 69 , could be partially cleaved by GluV8, because the residual group of Asn is relevant to that of Asp. To confirm this hypothesis, we compared the proteolytic activity of MCA peptides carrying various amino acids at their P1 position. The proteolytic activity of GluV8 was highest for the 2 peptide substrates carrying Glu at the P1 position and was significantly lower toward the 2 substrates carrying Asp at it (Table 3) .
Interestingly, GluV8 also possessed trace activity toward Z-Ala-Ala-Asn-MCA, which was at least 30-fold higher than those for residual MCA peptides that carried Ala, Phe, or Leu at their P1 position.
Furthermore, even unprocessed GluV8 possessed a proteolytic activity toward Ac-Ala-Ala-Asn-MCA.
At last, it should be noted that the C-terminal tri-peptide of the prosequence of GluV8, i.e.,
His 66 -Ala-Asn 68 , resembles the unprocessed-GluV8-cleavable substrate, Ac-Ala-Ala-Asn-MCA.
Discussion
We recently developed an E. coli expression system for GluV8 either by swapping the propeptide of it to that of GluSE or 4-amino acid substitutions from the amino acids of GluV8 to those of GluSE at equivalent positions, i.e., Glu62Gln, Glu65Ser, Ala67Pro, and Asn68Ser [13] . The present study demonstrated that the fifth substitution, i.e., Asp 36 of the propeptide to His, further enhanced the recovery of GluV8. This finding was surprising, because (i) the degradation activity of GluV8 for the Asp-X bond is substantially lower than that for the Glu-X bond (Table 3 ) [18] , and (ii) the position is located near the pre-pro border (Ala 29 -Leu 30 ) of GluV8; thereby, even if the Asp 36 -Asn 37 bond was hydrolyzed, the majority (82%) of the prosequence was still tagged to the mature moiety, and this form was enzymatically inactive [13] . However, this mutation actually improved the recovery, suggesting that the whole sequence of the propeptide is needed for the maintenance of the GluV8 proform. It has been reported that the prosequence of subtilisin is an intrinsically unstructured polypeptide that adopts an α-β structure only in the presence of the mature protease [19] . If this was also the case for GluV8, the deletion of a short segment from the prosequence may have a profound effect on its structure.
The substitutions of Ala 67 -Asn 68 to Pro-Ser additionally contributed to the enhancement of the expression of the recombinant protease. The proteolytic activity of non-processed GluV8 toward Z-Ala-Ala-Asn-MCA accounted 56% of that of processed GluV8. This finding indicates that the non-processed GluV8 could cleave the Asn 68 -Val 69 bond to some extent. It has been proposed that the α-amino group of the N-terminal residue is essential for the specific interaction with the γ-carboxyl group of Glu in a substrate peptide [20] . However, the protonation of the α-carboxyl group does not take an advantage in the association with amide group of Asn at the P1 position. This would explain the reason why the Asn-X bond was hydrolyzed either by the unprocessed or processed GluV8.
The P2-position amino acid of the maturation processing site of GluV8 was substituted from Ala 67
to Pro together with substitution of the P1 site from Asn 68 to Ser. It has been reported that Phe at P2 position was preferable for the GluV8-family endopeptidases and that Ala and Pro at P2 position were not [18] . Therefore, the substitution from Ala 67 to Pro may not affect the processing at the Ser 68 -Val 69 bond of GluV8. Nevertheless, we should point out that the X-Pro and Pro-X bonds are highly resistant to most proteases including GluV8, which might additively contribute to the prevention of proteolytic degradation, as GluV8 with short prosequences could exert a residual activity [13] .
The strategy developed for the expression of GluV8 in E. coli was successfully applied to the expression of another member of this family, GluSW. The N-terminal sequence of native GluSW was determined to Arg 64 -Ala-Asn-Val 67 , and this form is known to exert caseinolytic and glutamic acid-specific hydrolytic activities [10] . However, the present study clearly demonstrated that GluSW starting at Arg 64 had no proteolytic activity and that the hydrolysis of the Asn 66 -Val 67 bond was prerequisite for the maturation. Accordingly, we conclude that the processing mechanism of the GluV8 family proteases is commonly mediated by the cleavage at the Asn 68 -Val 69 bond of GluV8 and equivalent positions of GluSE and GluSW.
In conclusion, we established the E. coli expression system of glutamyl endopeptidases from S.
aureus and S. warneri. This strategy may shed light on the way for the expression of the proteases that were scarcely produced in E. coli to date. preprosequences of GluSE [9] , GluV8 [5] , GluSW [10] , and GluV8mut5 are aligned. Hyphens are deletions introduced for maximal matching. Amino acid numbers on the top are those for GluV8.
The border between of pre-and prosequences and that between pro-and mature sequences of GluV8 and GluSE are indicated by arrows. X indicates the proposed pro-mature border of GluSW [10] . The amino acids, Glu and Asp, which could be auto-proteolysed by glutamyl endopeptidases are underlined.
Asterisks indicate the 5 amino acids substituted in GluV8mut5. (b) Amino acid identity of pre-, pro-, and mature sequences among GluSE, GluV8, and GluSW. 

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