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Lethal toxin (LeTx) is a vital virulence factor of Bacillus anthracis and has been postulated to act as a Zn²⁺ protease mediating the fatal symptoms observed during anthrax infections by hyperstimulation of host macrophage inflammatory pathways (5, 6, 8, 10). LeTx is an A-B toxin comprised of two distinct proteins. Protective antigen (PA; 735 residues, 82.6 kDa) serves as the B moiety, directing binding to cellular membrane receptors and translocation of its catalytic partners into the cytoplasm (5, 11). Lethal factor (LF; 776 residues, 90.2 kDa) acts as the A moiety (5, 11). Evidence presented by Klimpel et al. demonstrates that LF is a zinc-binding protein which contains the HEXXH motif in its carboxy-terminal (activity) region at residues 686 to 690 (LF_686-690) (10). They hypothesized that LF requires zinc for activity and perhaps functions as a Zn²⁺-dependent protease, thus having functions similar to those of, and having an active-site motif in common with, the botulinum and tetanus neurotoxins, albeit with differing cell tropisms, target substrates, and disease sequellae (16). Here, we demonstrate LF-specific, Zn²⁺-dependent cleavage of synthetic peptides in vitro. These data, as well as those from protease inhibitor profiles, metal ion substitution studies, and mutational analysis of residues within LF_686-690 that arrest activity, strongly support LF as demonstrating the activities of a Zn²⁺-dependent neutral endoprotease.

Anthrax toxin purification. LF, PA, and mutants were purified either from B. anthracis Sterne or as recombinant proteins from Escherichia coli (6-8, 14). B. anthracis cultures were grown in defined RM toxin production medium (13). Culture supernatants were sterilized by passage through a 0.22-µm-pore-size filter (Millipore, Bedford, Mass.) and concentrated to 500 ml with the Minitan ultrafiltration system (Millipore). Ammonium sulfate was added to 75%, and the protein pellet was collected and suspended in 20 mM Tris-HCl (pH 8.0) and dialyzed extensively against the same buffer. Very efficient purification was performed by MonoQ anion-exchange fast-performance liquid chromatography (FPLC) (Pharmacia Biotech, Piscataway, N.J.). PA eluted at 130 to 140 mM NaCl, and LF eluted at 250 to 270 mM NaCl. The recombinant proteins expressed in pET15b are produced with an amino-terminal hexa-histidine tag, allowing purification by FPLC affinity chromatography on a HiTrap (Pharmacia Biotech) chelating column. Cultures of E. coli BLR(DE3)/pET15b-LF (or indicated LF mutants) were grown in Luria broth containing ampicillin (100 µg ml¹) to an optical density at 600 nm of 0.7 to 1.0, and expression was induced by the addition of IPTG (isopropyl--D-thiogalactopyranoside; 1 mM) for 12 h at 18°C. Cell lysates were prepared by French press, cleared by centrifugation, and injected via FPLC (Pharmacia Biotech) onto a HiTrap column charged with Ni²⁺. Recombinant histidine-tagged LF (wild type [wt] or mutant) eluted at approximately 100 mM imidazole. Eluted protein was further purified by gel filtration on a 320-ml Sephacryl-200 FPLC column. By these methods, PA and LF were each determined to be 95 to 99% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PA and LF proteins were assayed for cytoxicity activities by a standard ⁵¹Cr release assay of a sensitive macrophage cell line (RAW 264.7 ATCC TIB-71) (11, 13). Mutant proteins LF^E687C, LF^H686A, LF^H690A, and LF^H686A+H690A were obtained from Kurt Klimpel (10) as direct mutants of wt anthrax LF. LF^E687D was created as a recombinant mutant in E. coli BLR(DE3) by using the pET15b plasmid and purified as described above.

Protease assay conditions. Oligopeptides were obtained from Sigma. The reaction buffer used was 25 mM potassium phosphate (pH 7.0) containing 20 µM ZnSO₄ and 20 µM CaCl₂. Each reaction mixture contained as indicated from 250 µM to 1 mM substrate and 0.25 to 2.8 µM enzyme (23 to 250 µg/ml) in a final volume of 100 µl. After incubation at 37°C for 3 to 24 h (as indicated), the reactions were quenched with 1 µl of 10 M HCl and the mixtures were injected onto a C₁₈ HP Hypersil-octyldecyl silane column (100 by 4.6 mm, 120-Å pore size, 5-µm particle size) or a C₈ Rainin Microsorb-MV column (250 by 4.6 mm, 300-Å pore size, 5-µm particle size) with a Hewlett-Packard 1050 high-performance liquid chromatography (HPLC) system. The aqueous phase used with the C₁₈ column was 25 mM phosphate buffer (pH 7.5) with 2% (vol/vol) methanol (MeOH) and 2% (vol/vol) tetrahydrofuran, and the mobile phase was 100% MeOH. The aqueous phase used with the C₈ column was 0.1% (vol/vol) trifluoroacetic acid (TFA) in water, and the mobile phase was 0.1% (vol/vol) TFA in acetonitrile. Peptide peaks were detected by UV absorption at 215 and 274 nm. For further analysis by mass spectrometry or protein sequencing, peaks were collected as they eluted. For kinetic studies, reaction mixtures of 100 µM to 1 mM substrate with 250 nM LF were run at 25°C. Samples were injected into the HPLC at 3-h intervals. Velocities were then calculated by measuring the reduction of the starting substrate peak over time or by measuring the formation of product peaks over time. In inhibitor studies, enzyme and inhibitors were coincubated at room temperature for 1 h before addition of substrate. For studies with chelating inhibitors, there was no addition of exogenous metals. Otherwise, the conditions were the same as those described above. For pH studies, 250 ng of LF was added to 500 µM substrate in a buffer of 25 mM phosphate with pHs ranging from 5.8 to 8.2. Phosphate buffers were made from appropriate amounts of mono- and dibasic sodium phosphate. Reactions were analyzed after 4 h at 37°C as described above. Results indicated a pH optimum of approximately 6.75, with less than 5% activity at pH 5.5 (results not shown). Thus, the addition of HCl to reaction mixtures was determined to be the best way to quench reactions before HPLC analysis. To create apo-enzyme (no bound metals), purified LF was dialyzed against 10 mM EDTA-1 mM 1,10-phenanthroline for 24 h at 4°C. The proteins were then dialyzed against 20 mM Tris (pH 7.5) in ultrapure water (15 M), for 24 h at 4°C, with four buffer changes.

Peptide fragment analysis. Peptides purified by reverse-phase HPLC (RP-HPLC) were analyzed by time-of-flight mass spectrometry. The mass spectrum was recorded by using nitrocellulose targets (9) in an Applied Biosystems Bio-Ion plasma-desorption instrument. The spectrum was accumulated for 10⁶ fission events corresponding to approximately 10 min. Further details of the instrumentation and spectral analysis have been described elsewhere (19). Peptide analysis was kindly performed by the Duke Comprehensive Cancer Center Facility, directed by Jan J. Enghild.

Hydrolysis of synthetic peptides. To directly assess whether LF is capable of endopeptidase activity, synthetic peptides (or p-nitroanilide-derivatized peptides) as well as a variety of purified proteins were obtained as test substrates in an arbitrary manner. However, the lengths of the synthetic peptides ranged from 2 to 39 residues with efforts to vary both amino acid composition and primary sequence. The overwhelming majority of these substrates were not affected by LF in any discernible way, although they were cleaved by appropriate control proteases (e.g., trypsin, pronase, thermolysin) (data not shown). A complete list of substrates tested in this study is available upon request.

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View larger version (20K): [in this window] [in a new window]   FIG. 1.   HPLC elution profiles of synthetic peptides cleaved by anthrax LF. The dashed line represents 500 µM substrate alone without LF, and the solid black line represents substrate plus LF. The gray line indicates percent mobile phase. All reactions occurred at pH 7.0 in 25 mM phosphate buffer. (A) RP-HPLC elution profile of the reaction with peptide 1 (ELYENKPRRPYIL) and 23 ng of LF per ml after 18 h at 37°C. The HPLC running buffer contained 25 mM potassium phosphate (pH 7.5), 2% MeOH, and 2% tetrahydrofuran. (B) Elution profile of the reaction with peptide 2 (FGFLPIYRRPAS) and 250 ng of LF per ml after 4 h at 37°C. The HPLC running buffer was 0.1% TFA in water. The mobile phase was 0.1% TFA in acetonitrile. (C) Elution profile of the reaction with peptide 3 (IARRHPYFL) and 250 ng of LF per ml after 90 min at 37°C. The HPLC running buffer was 0.1% TFA in water. The mobile phase was 0.1% TFA in acetonitrile. All reactions were monitored by UV absorption at 215 nm (see text for more details). The HPLC flow rate was 1.0 ml/min. These results are typical examples of experiments repeated many times.

Inhibitor profiles. LF cleavage of peptides was completely inhibited by the addition of either 1 mM 1,10-phenanthroline or 10 mM EDTA, both of which chelate zinc (Table 2). Activity was partially inhibited by 5 µM EGTA. This indicates that certain metal ions are essential for LF activity in vitro. LF that was cleared of metals through dialysis with EDTA and phenanthroline (see Materials and Methods) showed no propensity to cleave peptides. As individual metals are added back to LF, it is clear that both zinc and calcium are essential for full protease activity (Fig. 2). Additionally, specific amino acid hydroxamates, selective inhibitors of zinc metalloproteases, have been shown to inhibit LF both in vitro and in vivo. Amino acid hydroxamates are reversible inhibitors that fit the enzyme's active site while chelating the zinc ion (3). Tyrosine and leucine hydroxamate showed the best in vitro inhibition with 50% inhibitory concentrations of approximately 300 and 350 µM, respectively. Additionally, ZINCOV, a novel hydroxamate, inhibits LF in vitro protease activity at 350 µM and completely protects macrophages in vivo at a concentration of 500 µM (in vivo data not shown). The ability of hydroxamates to inhibit LF supports LF having zinc metalloprotease activity. Further, the specific ability of tyrosine and leucine hydroxamates to inhibit LF may implicate these amino acids as important for active site binding. Previously, relatively high concentrations (200 µM) of the protease inhibitors bestatin and lysine CMK were shown to protect cultured macrophages from lysis by anthrax LeTx (10). In contrast, bestatin and lysine CMK did not inhibit in vitro LF proteolysis of peptides 1 and 2 at concentrations ranging from 50 µM to 1 mM (Table 2), suggesting that the protective effect of these inhibitors observed with LF-challenged cultured cells might not be due to direct inhibition of the toxin's enzymatic activity but rather to some other event in the cytolytic cascade. As an example of this phenomenon, in lipopolysaccharide-stimulated monocytes, inhibitors of metalloproteases were observed to block maturation and release of shock-inducing cytokines (15). It is possible that bestatin and lysine CMK inhibit an event downstream from initial target cleavage by LF. Other classes of protease inhibitors, such as phenylmethylsulfonyl fluoride, N-succinyl-L-proline, tosyl lysine CMK, tosyl phenylalanine CMK, and nitrobestatin, did not inhibit LF activity in vitro (Table 2).

View larger version (33K): [in this window] [in a new window]   FIG. 2.   In vitro protease activity of metal-reconstituted anthrax LF. Comparison of the areas of the major product peaks (see Fig. 1A, peak labeled ELYENKPRRP) created from the reaction with peptide 1 (ELYENKPRRPYIL) and LF. Reactions of 50 ng of LF per ml with 1 mM ELYENKPRRPYIL occurred at pH 7.0 in 25 mM potassium phosphate buffer containing a 100 µM concentration of the indicated metal(s). Reactions were monitored as described in the text. All metals were chloride salts, except for zinc and nickel which were sulfate salts. After 9 h at 37°C, reaction mixtures were injected into the HPLC. It is important to note that the first three columns indicate the need for both zinc and calcium ions for full catalysis. No reaction was ever observed in the absence of metals, or with calcium alone, cobalt alone, magnesium alone, manganese alone, or nickel alone. The fastest reaction consistently was that in which only zinc and calcium were added. These results are an average of two experiments.

Active-site mutations. Amino acid substitutions in LF_686-690 (the HEXXH motif) were previously shown to be incapable of killing cultured macrophages, suggesting that this region is important for cytotoxicity and perhaps is involved in catalytic function (10). To determine whether this consensus thermolysin-like active-site motif may be directly involved with cleavage of peptides, and to ensure that all hydrolytic activities observed in our assays are specific to LF, amino acid substitutions in LF_686-690 were tested for the ability to cleave peptides 1 and 2. Mutant toxin molecules assayed were LF^E687C, LF^E687D, LF^H686A, LF^H690A, and LF^H686A+H690A. The recombinant mutants proteins were purified as stable, full-length molecules as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with high-titer polyclonal antitoxin. All histidine mutants were found to have decreased zinc binding as compared to that of wt LF and were unable to kill macrophages in LeTx assays (10; also data not shown). All mutants were mixed individually with peptide 1, 2, or 3 to determine hydrolytic activity. No significant reduction in substrate concentration or generation of cleavage product was observed by RP-HPLC even after 18 h at 37°C (Table 3). This indicates that LF^H686 and LF^H690 are important residues for both Zn²⁺ binding and catalysis as well as cytotoxicity. The LF^E687C mutant was found to bind zinc at a level equal to that of wt LF yet was also unable to kill cultured cells in LeTx assays (10; also data not shown). However, as with the histidine mutants, LF^E687C showed no observable cleavage of peptide 1 even after incubation for 18 h at 37°C (Table 3). Additionally, work with our mutant LF^E687D showed no ability to cleave peptides in vitro, even at a very high concentration of enzyme. This mutant was also unable to kill macrophages in vivo (data not shown). This indicates that LF^E687 is important for cytotoxicity and catalysis but not for Zn²⁺ binding. Importantly, the loss of hydrolytic activities associated with point mutations in the active-site region acts as a control for assignment of hydrolytic functions to LF and not to some undefined contaminating protease.

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Known LF domains. The first 255 amino acids are involved in binding to PA, and this region has been termed LFn. This region has a high degree of homology to the first 255 amino acids of anthrax edema factor, which also binds PA. Residues 315 to 416 contain five repeat regions that follow the consensus sequence for two EF-hand calcium-binding motifs (e.g., calmodulin). Residues 686 to 690 contain a thermolysin-like zinc metalloprotease motif HEXXH. Additionally, residues 745 to 749 (HSTDH), which are similar to the inverted zinc metalloprotease motif HXXEH (e.g., insulin-degrading enzyme), could potentially act as another zinc site, either structural or enzymatic.