Substrate Cleavage Profiling Suggests a Distinct Function of Bacteroides fragilis Metalloproteinases (Fragilysin and Metalloproteinase II) at the Microbiome-Inflammation-Cancer Interface*

Background: Two distinct metalloproteinase types (fragilysin and metalloproteinase II/MPII) are encoded by the Bacteroides fragilis pathogenicity island. Results: Our assays determined substrate cleavage characteristics of fragilysin and MPII. Conclusion: MPII is the first zinc metalloproteinase with the dibasic cleavage preferences. Significance: Our results are important for understanding B. fragilis virulence and fundamental roles of the microbiome in human health and disease. Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.

Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg2Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)2) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.
A variety of microbial communities (the microbiome) exists in the human body, playing fundamental roles in human health and disease (1,2). Normally, bacteria outnumber human cells within an individual by at least an order of magnitude. The gastrointestinal tract is part of the direct interface between the human organism and the microbiome. In certain circumstances, the beneficial relationship between the microbiome and the gastrointestinal tract becomes disrupted, causing disturbances leading to disease.
Chronic inflammation affects all phases of carcinogenesis, from favoring the initial genetic alterations that drive cancer formation to acting as a tumor promoter by establishing conditions in the surrounding tissues that allow the tumor to progress and metastasize (3)(4)(5)(6). For example, chronic hepatitis B or C virus infections frequently lead to liver cancer (7), and chronic Helicobacter pylori infection leads to gastric cancer in some patients (8 -10). Increased cancer incidence is likewise found in experimental mouse models of both infection-induced and noninfectious inflammation (11,12).
There is a consensus among researches that metalloproteinase activity is essential for B. fragilis virulence and that this activity is encoded by the 6-kb pathogenicity island in enterotoxigenic B. fragilis strains (14,21,26,27). The island contains at least two metalloproteinase genes. These genes encode fragilysin (FRA; also termed B. fragilis toxin or BFT), demonstrated to be important to pathogenesis, and metalloproteinase II (MPII), also a potential virulence protein. FRA exists in three homologous isoforms (FRA1, -2, and -3) with the sequence identity over 90%. In turn, sequence identity between FRAs and MPII is only 25% (Fig. 1).
FRAs and MPII are secretory zinc metalloproteinases with a zinc-binding HEXXHXXGXXH motif and a characteristic Met turn. These structural features, especially when combined, indicate that both FRAs and MPII exhibit the matrix metalloproteinases (MMP)/a disintegrin and metalloproteinase (ADAM) fold (28 -31). The overall level of homology between the catalytic domain of bacterial FRAs and MPII and mammalian MMPs/ADAMs, however, is low.
Although there is limited information about the structuralfunctional features of FRAs (17,32), the biochemical characteristics of MPII remain completely unknown. Because MPII has not yet been characterized, it is unclear if MPII can facilitate the toxigenic effect of FRAs in causing diarrhea, inflammatory bowel disease, and CRC (14,26,30,33,34). As a result, we cannot decipher, at the molecular level, how the proteolytic activity of B. fragilis tailors the normal luminal epithelium for inflammation and disease onset. Understanding the substrate cleavage specificity of MPII relative to FRAs may help to determine how infection-associated inflammation enhances carcinogenesis in the affected organs and how we may find a means to fight the disease.
Here, we performed a comparative characterization of MPII and FRA3, a representative of the FRA isoforms. Our data imply that, in contrast with the FRA family members, the unconven-tional MPII cleavage preferences mimic those of furin-like proprotein convertases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on our results, we suggest that the combined (rather than individual) activity of MPII and FRAs is required for B. fragilis virulence.
Detection of B. fragilis DNA in Patient Samples-The frozen tumor and matching normal tissue deidentified biopsies were obtained from our preexisting collection of proximal CRC cancer specimens. Genomic DNA was extracted from the tissue samples using the DNeasy blood and tissue DNA purification system (Qiagen). The 501-bp fragment of the B. fragilis 16 S rRNA gene was amplified in the 100-l PCRs containing genomic DNA (100 ng), the forward and reverse primers (5Ј-ATAGCCTTTCGAAAGRAAGAT-3Ј and 5Ј-CCAGTATCA-ACTGCAATTTTA-3, respectively; 0.3 M each), Crimson Taq DNA polymerase (1 unit), and 12.5 mM Tricine buffer, pH 8.5, supplemented with 42.5 mM KCl, 1.5 mM MgCl 2 , 6% dextran, and 0.2 mM dNTP mix. DNA amplifications were performed using denaturing of the samples at 95°C for 5 min followed by 35 PCR amplifications (95°C for 30 s, 52°C for 30 s, 72°C for 1 min). The products were separated by 2% agarose gel electrophoresis. Amplified 501-bp products were purified and sequenced to confirm their authenticity and identity. Twosided Fisher's exact test was used to evaluate the statistical significance of the association of the bacteria with colorectal cancer.
Cloning, Expression, and Purification of MPII and FRA3-The full-length cDNA coding for the wild type MPII proenzyme (gi:3046922) and the FRA3 proenzyme (PDB accession code 3P24; gi:315583580) were synthesized by Genewiz. PCR with the 5Ј-CACCATGCACCATCACCATCACCATGGAGCCT-GTGCCGATGACCTG-3Ј and 5Ј-TCAATGGTGGTGATG-GTGGTGCTTGTCATCGTCATCTTTGTAGTCCTTTTG-GATGCACTCCAG-3Ј oligonucleotides as the forward and reverse primers, respectively, was then used to insert the His 6 tags (both N-and C-terminally) and the FLAG tag (C-terminally) into the MPII template. Similarly, the 5Ј-CACCATGCA-CCATCACCATCACCATGGAGCCTGCAGCAATGAGGC-C-3Ј and 5Ј-TCAATGGTGGTGATGGTGGTGCTTGTCAT-CGTCATCTTTGTAGTCACCATCTGCGATCTCCCAGC-C-3Ј as the forward and reverse primers, respectively, were used to incorporate two His 6 tags and a single FLAG tag into the FRA3 construct.
The MPII E352A proteolytically inactive mutant, in which Ala replaced the catalytically essential Glu residue of the active site, was constructed using PCR mutagenesis with 5Ј-CCGT-ATACACTGGCACACGCGATCGGCCATCTGCTGGGC-3Ј and 5Ј-GCCCAGCAGATGGCCGATCGCGTGTGCCAGT-GTATACGG-3Ј as the forward and reverse primers, respectively, and the wild type MPII template (mutant nucleotides are underlined). Similarly, 5Ј-GATGGCCCACGCACTGGGCCA-CATC-3Ј and 5Ј-GATGTGGCCCAGTGCGTGGGCCAT-C-3Ј as the forward and reverse primers, respectively, and the wild type FRA3 template were used to generate the FRA3 E349A inactive mutant (mutant nucleotides are underlined). The authenticity of the constructs was confirmed by DNA sequencing.
The wild type and mutant tagged constructs were recloned into the pET101 expression vector (Invitrogen). The recombinant pET101 plasmids were used to transform competent E. coli BL21 (DE3) Codon Plus cells (Stratagene). Transformed cells were grown at 30°C in LB broth containing ampicillin (0.1 mg/ml). Cultures were induced with 0.6 mM IPTG for 16 h at 18°C. Cells were collected by centrifugation, resuspended in Tris-HCl buffer, pH 8.0, containing 1 M NaCl, and disrupted by sonication. The pellet was removed by centrifugation (40,000 ϫ g; 30 min). The constructs were then purified from the supernatant fraction on a Co 2ϩ -chelating Sepharose Fast Flow column (GE Healthcare), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, supplemented with 1 M NaCl. After washing out the impurities using the same buffer supplemented with 35 mM imidazole, the bound material was eluted using a 35-500 mM gradient of imidazole. The FRA fractions were combined and dialyzed against 20 mM Tris-HCl, pH 8.0, containing 150 mM NaCl. The purified material was kept at Ϫ80°C until use. The purity of the material was tested by SDS-gel electrophoresis (12% NuPAGE-MOPS, Invitrogen) followed by Coomassie staining and by Western blotting with an anti-FLAG tag antibody.
Activation of MPII and FRA3-The MPII E352A and FRA3 constructs (3 g; 3 M each) were co-incubated in PBS for 1 h at 37°C with increasing concentrations of trypsin. Similarly, FRA3 (3 g; 3 M) was co-incubated for 1 h at 37°C with increasing concentrations of active MPII in 50 mM HEPES buffer, pH 8.0, containing 1 mM CaCl 2 and 50 M ZnCl 2 . The cleavage was stopped by adding a 5ϫ SDS sample buffer to the reactions. The digested samples were analyzed by SDS-gel electrophoresis in 12% NuPAGE-MOPS gels.
Multiplex Peptide Cleavage Assay-Peptide pool preparation and the following in vitro cleavage assays were performed using a methodology similar to those described in our recent publications (36 -38). Template DNA encoding 10-amino acid residue-long peptide substrates was transcribed in vitro to produce the corresponding mRNAs. In vitro translation was then used to generate peptides covalently attached to their mRNA templates (39). To increase their stability, the peptide-mRNA fusions were converted to the corresponding covalent peptide-cDNA fusions (40). The peptide-cDNA fusions were biotinylated at the N terminus and immobilized on streptavidin-coated magnetic beads. We also spiked six biotinylated oligonucleotides into the peptide-cDNA pool to use as internal standards for normalization after PCRs. The magnetic beads with the immobilized peptide-cDNA pool (2 pmol) were co-incubated with MPII and FRA3 (1.2 or 12 pmol) at 37°C for 30 min in 3-l reactions containing 50 mM HEPES, pH 6.8, 10 mM CaCl 2 , and 10 M ZnCl 2 . Reactions without the proteinases were used as negative controls. To identify cleaved peptide substrates, the cDNA molecules released by the proteinase treatment were collected, the DNA adapters required for sequencing were installed by PCR (41), and the obtained DNA constructs were sequenced using a MiSeq sequencing instrument (Illumina). In this study, we produced and tested 316 peptide sequences.
Peptide abundance in solution was quantified by counts of DNA reads corresponding to each peptide sequence. The cleavage efficiencies were determined by comparing counts in the proteinase-treated samples versus proteinase-less, buffer-only controls (each in triplicate). Next, we normalized the data for each of six different biotinylated substrates (internal standard) across the whole data set. In order to calculate an enrichment ratio for each substrate, we considered counts (in triplicate) in a positive sample over the average of counts of a control sample (buffer-only, no proteinase) ϩ 3 ϫ S.D. An enrichment ratio (ER) ϭ 1 indicates that counts in a positive sample are 3 ϫ S.D. values above the "buffer-only, no proteinase" negative sample.
Mass Sequence Logos-The sequence logos were obtained by calculating cleavage efficiency over the entire set of substrates and then selecting substrates with an ER value of Ͼ3. These substrates were considered susceptible to proteolysis. Substrates with an ER value of Ͻ3 were considered resistant to proteolysis. The resulting logos were created by the Web-based IceLogo program (42).
Molecular Modeling of MPII, FRA1, FRA2, and FRA3-Molecular modeling of the substrate complexes of MPII and FRA3 was initiated from the crystal structure of FRA3 (PDB entry 3P24) (30). In this structure, the ␤11 strand of the FRA3 prodomain directly interacts with and tightly fits into the active site of the catalytic domain. The strand, however, runs in the opposite orientation relative to the substrate. To model the PRPLRAWGAA substrate, the Leu 186 -Val 201 segment of the ␤11 strand was deleted in silico. The substrate was then modeled by placing the substrate's side chain heavy atoms at the positions of the heavy atoms in the ␤11 strand. The further optimization of the main and side chain atoms of the substrate was performed using molecular mechanical minimization and short molecular dynamics simulations via the Amber12 modeling package and FF99SB force field and the generalized Born method for representing the solution environment implicitly (43)(44)(45)(46). In the course of our molecular mechanical optimization procedure, the proper orientation of the carbonyl group of the scissile bond relative to the active site zinc ion was maintained, and acetyl and N-methyl groups for capping the N and C termini of the modeled substrate were used. The structure of MPII was prepared by homology modeling using PDB entry 3P24 as a template and FFAS03 (47,48) and Modeler (49) software. The fold of the PGRLR2RSGAA substrate mimics the fold of the PRPL-R2AWGAA substrate in the FRA3 structure. The structures of FRA1 and FRA2 were modeled using FRA3 (PDB entry 3P24) as a template and Modeler software. The final models were displayed using PyMOL (Schrödinger, LLC, New York).

Activity Assays with the Fluorescence-quenched Peptide-
The assay for MPII cleavage activity was performed in 50 mM HEPES buffer, pH 8.0, containing 1 mM CaCl 2 , 0.5 mM MgCl 2 , and 10 M ZnCl 2 . The 5-FAM-SLGRKIQIQK(QXL520)-NH 2 substrate and enzyme concentrations were 10 M and 50 nM, respectively. The total assay volume was 0.2 ml. Initial reaction velocities were monitored continuously at ex ϭ 488 nm and em ϭ 520 nm using a Spectramax Gemini EM fluorescence spectrophotometer (Molecular Devices). All assays were performed in triplicate in wells of a 96-well plate.
Where indicated, MPII (50 nM) was co-incubated for 30 min at 20°C with increasing concentrations of natural tissue inhibitors of MMPs (TIMP-1, TIMP-2, and TIMP-3) and small molecule hydroxamate inhibitors of MMPs (GM6001/Ilomastat, BB94/Batimastat, and AG3340/Prinomastat). The residual activity of the samples was then measured as above. EC 50 values were calculated by determining the concentrations of TIMPs and hydroxamate inhibitors needed to inhibit 50% of the MPII cleavage activity. GraphPad Prism was used as fitting software.
MPII Proteolysis of Proteins in Vitro-Anthrax PA83 (2 g; ϳ1 M) was co-incubated for 1 h at 37°C with increasing concentrations of MPII in 50 mM HEPES, pH 8.0, containing 1 mM CaCl 2 , 0.5 mM MgCl 2 , and 10 M ZnCl 2 . The total volume of the reactions was 20 l. Where indicated, GM6001 (1 M) was added to the reactions to inhibit MPII. The cleavage reaction was stopped by adding a 5ϫ SDS sample buffer. The digest samples were analyzed by SDS-PAGE using 4 -20% polyacrylamide gel.

B. fragilis in
Human CRC-To test if B. fragilis is more prevalent in the CRC tumor biopsies as compared with the matching normal tissue, we isolated the total genomic DNA from 30 tumor biopsies and 30 matching normal tissue specimens. We then amplified by PCR the 501-bp fragment of the B. fragilis 16 S RNA gene using the isolated DNA samples. Selected amplified bands were sequenced to confirm their B. fragilis identity. Our data clearly demonstrated the presence of the B. fragilis 16 S RNA in 22 of 30 tumor biopsies (ϳ73%) but only in 13 of 30 matching normal tissue samples (ϳ43%), thus suggesting a statistically significant association of the bacteria with CRC (Fig.  2). In addition, the bacterium was found only in those matching normal tissues, which were derived from the patients with the B. fragilis-positive tumor samples.
Recombinant Constructs of MPII and FRA3-To determine the potential proinflammatory, cancer-promoting role of B. fragilis toxin, we successfully cloned the full-length genes coding for B. fragilis MPII and FRA3. To facilitate their purification, the constructs were flanked by an N-terminal and C-terminal His 6 tag and, additionally, by a C-terminal FLAG tag. We also constructed the catalytically inactive MPII and FRA3 (E352A and E349A mutants, respectively) in which the catalytically essential Glu residue of the active site was mutated into Ala. The constructs were expressed in and purified from lysates of E. coli (Fig. 3). MPII was readily self-activated, whereas the FRA3 proform was noticeably more stable, and only a minor fraction of the active enzyme accumulated in the purified FRA3 samples during storage. As judged by the mobility of the digest products in SDS gels and in agreement with the observations by others (30), trypsin proteolysis converted the FRA3 proenzyme and the MPII E352A inert mutant into the mature proteinase (data not shown). In turn, MPII was unable to convert the FRA3 proenzyme into the active proteinase (data not shown), suggesting that either human trypsin or an unidentified bacterial proteinase activates FRA3 in the gut in vivo.
Multiplex Cleavage Assay of MPII and FRA3-In order to quickly characterize the substrate preferences of the FRA cleavage, we used recently published technology for screening of customizable peptide pools. The utility of peptide-cDNA fusion pools in multiplexed assays was demonstrated in our previous reports using multiple proteinases, including human furin, hepatitis C virus NS3/4A proteinase, and thrombin (36 -38).
The design of the set of 316 10-mer peptides used in the assay was based on the known cleavage site of FRA3 in E-cadherin (17,21) and on the consensus cleavage sites (Pro-X-X2Leu) of human MMPs. The original cleavage data are presented in supplemental Table S1 and summarized in Table 1. To determine the position of scissile bonds, 11 representative individual peptides were resynthesized using standard peptide synthesis methodology. The individual peptides were then digested by The arrows indicate the cleavages at the prodomain excision sites in the proform. These cleavages release the prodomain and the mature protease. SP, PRO, and CAT, signal peptide, prodomain, and catalytic domain, respectively. Right, recombinant MPII and FRA3 tagged with the His 6 and FLAG tags. E352A and E349A indicate the catalytically inactive mutants of MPII and FRA3, respectively, in which the catalytically essential Glu residue of the active site was mutated into Ala. B, SDS-gel electrophoresis of the purified constructs. Note that the wild-type MPII is readily self-activated, whereas the wild-type FRA3 is significantly more stable in its proenzyme form during storage. CS, Coomassie staining; WB, Western blotting with an anti-FLAG tag antibody.

TABLE 1 Peptide sequences efficiently cleaved by MPII and FRA3
High numbers indicate a high cleavage efficiency of the peptides in the multiplexed cleavage assay. MS of the digest reactions was used to identify the mass of the cleavage fragments and, consequently, the scissile bonds (arrow). Enrichment ratio (ER) is a measure of cleavage activity.
MPII and FRA3. The digest reactions were analyzed by MALDI-TOF mass spectrometry to identify resulting digest fragments and, consequently, the scissile bonds. The MS data directly correlated with the results of the multiplex assay. The representative mass spectrometry analysis data are shown in Fig. 4A.
Of 316 tested peptide sequences, Ͼ80 and Ͼ230 were cleaved by MPII and FRA3, respectively (ER Ͼ 3). In order to identify the most probable cleavage sequences, we included only peptides with ER Ͼ 3 in our analysis. Although the peptide set used in the experiments was limited in size, it was sufficient to show a clear difference in substrate preferences of MPII versus FRA3 (Fig. 4B). Our data imply that MPII prefers Arg at both the P1 and P1Ј positions. The presence of Leu at the P2 position, Arg or Gly at both the P3 and P3Ј positions, and Gly at the P4 position are all characteristic of substrates efficiently cleaved by MPII. In turn, FRA3 prefers the presence of Leu and Pro at the P2 and P5 positions, respectively. Arg/Ala, Pro/Gly/ Ser, and Arg/Ala/Leu are well tolerated at the P4, P3, and P1 positions, respectively. Overall, the resulting cleavage signature of FRA3 is most similar to the Pro-X-X-Leu consensus cleavage motif of MMPs.
Overall, our data suggest that there is a level of similarity between the dibasic cleavage signatures of MPII and the furinlike proprotein convertases (50). In agreement with this suggestion, anthrax PA83 treated with MPII appeared to be cleaved at the Arg 193 -Lys-Lys-Arg 196 furin cleavage motif. As a result, MPII transformed PA83, albeit less efficiently than furin, into the C-terminal PA63 and the N-terminal PA20 fragments (Fig.  4C).
In Vitro Cleavage Assay of MPII Activity-Our cleavage studies allowed us to elucidate a cleavage signature of MPII and FRA3 and thus to identify the commercially available fluorescence-quenched peptide 5-FAM-SLGRK2IQIQK(QXL520)-NH 2 as a substrate for MPII. This peptide employs a 5-FAM/ QXL520 FRET pair for quantification of enzyme activity, providing a convenient direct assay for MPII activity. As we expected based on the multiplex assay results, this peptide is cleaved by MPII but not by FRA3. The availability of this peptide substrate allowed us to readily establish the fundamental enzymological features of the MPII activity, including its pH and temperature optimum dependence and effects of salts and glycerol (Fig. 5). Our results imply that MPII is a thermolabile protease with pH optimum at pH 8.0, that MPII is fairly resistant to high salt concentrations, and that the MPII activity is not enhanced by glycerol, which is normally used for enzyme stabilization and activity enhancement. Because we were not able to locate any commercially available fluorescent quenched peptide substrates for FRA3, we could not perform the similar enzymological studies with FRA3. Overall, we conclude that the cleavage preferences we determined for FRA3 roughly represent those of the entire FRA subfamily and that the cleavage preferences of MPII and FRAs are distinct. Middle and bottom, AGLRRAALGG and SGHMHAALTA, respectively, were co-incubated with FRA3. The spectra of the digest, the intact peptide, the enzyme alone, and the buffer alone are shown by red, black, green, and magenta lines, respectively. AU, arbitrary units. B, frequency plot of the cleavage sequences of MPII and FRA3 in an IceLogo format. The height of a character is proportional to the frequency of the amino acid residue at the individual position of the cleaved peptide and is normalized for the amino acid encoded in the entire human genome according to RefSeq. C, MPII cleaves anthrax PA83. MPII and furin were co-incubated with PA83 at the indicated enzyme-substrate molar ratio. The resulting PA63 and PA20 are shown by arrows. The digests were separated by SDS-gel electrophoresis. Where indicated, GM6001 was added to the reactions. (51,52). In addition, small molecule inhibitors of a hydroxamate class are also readily available for the MMP/ ADAM studies in vitro and in cell-based tests (53)(54)(55)(56). These hydroxamates chelate the active site zinc atom and inactivate MMPs/ADAMs. Using the activity assay conditions we established, we identified compounds that inhibit MPII (Fig. 5C). The GM6001 hydroxamate inhibitor (N-((2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; potent wide-range inhibitor of multiple individual MMPs/ADAMs; known also as Galardin and Ilomastat) (57-59) was capable of inhibiting MPII (EC 50 ϭ 31 nM). However, GM6001 is ϳ100-fold more potent in inhibiting MMPs (e.g. MMP-2 with an EC 50 ϭ 0.4 nM) ( Table 2). Additional potent, broad spectrum hydroxamate inhibitors, including AG3340/ Prinomastat (3(S)-2,2-dimethyl-4-(4-pyridin-4-yloxy)-benzenesulfonyl)-thimorpholine-3-carboxylic acid hydroxyamide) and FIGURE 5. Biochemical characteristics and inhibitors of MPII. The cleavage activity of MPII was measured using 5-FAM-SLGRKIQIQK(QXL520)-NH 2 as a fluorescence-quenched peptide substrate. A-D, the effect of buffer pH, glycerol concentration, salt concentration, and temperature on the MPII cleavage activity, respectively. E, selected inhibitors of MPII; representative dose-response curves. The EC 50 values of TIMP-2 and the hydroxamate inhibitors (GM6001 and AG3340) were measured using 5-FAM-SLGRKIQIQK(QXL520)-NH 2 as a fluorescence-quenched peptide substrate.

Inhibitor
It becomes evident from this modeling that a large size S1Ј pocket can accommodate both small and large, hydrophobic and hydrophilic, and positively charged residues (but not the negatively charged Asp/Glu residues). This structural feature of the catalytic groove could explain a broad range specificity of FRA3 at the P1Ј position. The S1Ј pocket of FRA3 is organized by the side chains of Leu 314 , Gly 344 , Thr 371 , and Tyr 370 and by the backbone of Leu 365 and Tyr 367 . The less sizable, charged S1Ј pocket of MPII is probably organized by the side chains of Ser 319 , Tyr 347 , Tyr 373 , and Ser 374 and by the backbone of Ile 321 and Leu 368 . Arg of the peptide substrate (and, probably, Lys as well) matches a highly negatively charged S1Ј pocket of MPII. The presence of Glu 276 in S1 of MPII (versus Ala 276 in the corresponding position of FRA3) explains the preference of MPII (but not FRA3) for the P1 Arg. This preference, however, does exclude hydrolysis of the substrates with P1Ј residues distinct from Arg, such as Gly and Ala, both of which are accepted, albeit less efficiently, by MPII (supplemental Table S1). Selfactivation of MPII at the LSSR2A site provides an additional support for the above suggestion. The extended S2 site readily accommodates the hydrophobic Leu substrate residue in both MPII and FRA3. The 2.8 Å resolution x-ray crystal structure of MPII we recently solved correlates very closely with our modeled MPII structure, thus corroborating our modeling studies. 3 Because of the high sequence homology among FRA1, FRA2, and FRA3, the modeled structures of FRA1 and FRA2 are highly similar to that of FRA3 (PDB entry 3P24). There are a very few substitutions in FRA1 and especially in FRA2 that may affect their substrate binding mode and cleavage preferences as compared with those of FRA3 (Fig. 6B). Thus, the presence of Asp 320 and Arg 357 in FRA2 (versus Asn 320 and Asn 357 in FRA3) may have an insignificant effect on the P2 and P4 subsite specificity. In a similar way, the presence of Lys 312 , Met 316 , Glu 357 , Lys 277 , and Phe 319 in FRA1 (versus Asn 312 , Ile 316 , Asn 357 , Asp 277 , and Leu 319 in FRA3) may have a limited impact on the promiscuous P1-P4 and P2Ј subsites that accept multiple residue types in FRA3 (Fig. 4).

DISCUSSION
The determination of the substrate recognition specificity of a protease is a necessary first step in developing druglike antagonists. Knowledge of an optimal peptide substrate greatly facilitates many of the subsequent steps in drug development and, in some instances, can provide a lead that is usually a few steps from a drug.
Here, we, for the first time, extensively characterized the purified recombinant MPII and FRA3, a representative isoform of the FRA subfamily. Based on the results of the high throughput multiplexed cleavage assay we recently developed (36 -38), followed by digest of the individual selected peptides combined with the mass spectrometry analysis of the cleavage reactions, we revealed that MPII strongly prefers Arg residues at both the P1 and P1Ј positions. The presence of Leu at the P2 position, Arg or Gly at both the P3 and P3Ј positions, and Gly at the P4 position is also characteristic of the efficient substrates of MPII. From these perspectives, MPII mimics the dibasic Arg2Arg cleavage motif of the furin-like proprotein convertases. We suspect that MPII interferes with the processing of those membrane and soluble precursor proteins, which are incompletely processed by proprotein convertases. As a result, MPII proteolysis may result in an abnormal precursor/mature protein ratio for the multiple targets of proprotein convertase processing, leading to pathological consequences (e.g. via aberrations of the furin-regulated functions, including cell-to-cell signaling, cell movement, Rho and Notch signaling, and many others) (37). Intriguingly, MPII appears to be the first zinc metalloproteinase with a dibasic cleavage preference, suggesting a high level of versatility of metalloproteinase proteolysis. Unfortunately, the readily available pyroglutamic acid-Arg-Thr-Lys-Arg-methyl-coumaryl-7-amide and other similar furin substrates cannot be used with MPII because the Argmethyl-coumaryl-7-amide cleavage product is as quenched as the intact substrate.
In turn, FRA3 prefers the presence of Leu and Pro at the P2 and P5 positions of the substrate peptide, respectively. Arg/Ala, Pro/Gly/Ser, and Arg/Ala/Leu are well tolerated at the P4, P3, and P1, respectively. Overall, the cleavage signature of FRA3 is related to the Pro-X-X-Leu consensus cleavage motif of MMPs. Based on the recently published crystal structure of FRA3 (PDB entry 3P24) and the follow-on our in silico modeling of the MPII structure, we provided structural evidence for the specificity of MPII and FRA3. Our efforts identified the commercially available fluorescence-quenched peptide 5-FAM-SLGRK2IQIQK(QXL520)-NH 2 as a substrate for the routine cleavage studies of MPII. This substrate is efficiently cleaved by MPII but not by FRA3, for which a commercially available peptide substrate is currently unavailable. Our results, however, should allow us to develop FRA3 substrates to facilitate laboratory studies of the toxin isoforms.
We appreciate that many proteases harbor exosites that can be important elements for substrate selectivity and specificity. Likewise, structural properties of the substrate can be relevant for proper exposure of cleavage sites. Naturally, the use of short The molecular surface of MPII and FRA3 with the labeled substrate subsites is colored according to the electrostatic potential (red, blue, and white represent negative, positive, and neutral electrostatic potential values, respectively). The active site zinc ion is shown as a gray sphere. B, modeled structures of FRA1 and FRA2 versus FRA3 (PDB entry 3P24). The modeled PRPLR2AWGAA substrate is shown as green sticks. The structures of the mature FRA1, FRA2, and FRA3 are shown as gray schematics in the background and as transparent electrostatic potential surfaces (red, blue, and white represent negative, positive, and neutral electrostatic potential values, respectively). The arrows point to the residue positions that may affect substrate binding and that are distinct in FRA1 and FRA2 versus FRA3. peptides in protease screenings is hampered by the lack of this structural information. Nevertheless, our results represent the first step in the right direction, and cell-based assays in the search for natural substrates of MPII in colon epithelial cells will follow shortly.
Our data suggest that because of their inefficient binding capacity, TIMPs, natural inhibitors of MMPs, cannot neutralize the FRA activity in the course of B. fragilis infection ( Table 2). More efficient inhibitors might limit FRA activity in a clinically beneficial manner. Therefore, peptide substrates that mimic the cleavage preference of the FRA isoforms are urgently required for inhibitor design and the follow-on inhibitory studies.
In sum, our results may help to determine, at the molecular level, generalized mechanisms by which the microbiota contributes to inflammation and increases cancer risk in infected patients (70). Our results may be useful in the development of tools to monitor the presence of bacterial toxigenic proteinases and the bacterium itself in infected patients. Such tools may help to determine where, how, and when B. fragilis fragilysins contribute to infection, to validate the value of novel inhibitors of these proteinases, and, last, to control these proteinases in a manner that is clinically beneficial for the infected patients.