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. 2015 Nov 6;290(45):27248-60.
doi: 10.1074/jbc.M115.648782. Epub 2015 Sep 18.

Calcium Regulates the Activity and Structural Stability of Tpr, a Bacterial Calpain-like Peptidase

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Free PMC article

Calcium Regulates the Activity and Structural Stability of Tpr, a Bacterial Calpain-like Peptidase

Dominika Staniec et al. J Biol Chem. .
Free PMC article

Abstract

Porphyromonas gingivalis is a peptide-fermenting asaccharolytic periodontal pathogen. Its genome contains several genes encoding cysteine peptidases other than gingipains. One of these genes (PG1055) encodes a protein called Tpr (thiol protease) that has sequence similarity to cysteine peptidases of the papain and calpain families. In this study we biochemically characterize Tpr. We found that the 55-kDa Tpr inactive zymogen proteolytically processes itself into active forms of 48, 37, and 33 kDa via sequential truncations at the N terminus. These processed molecular forms of Tpr are associated with the bacterial outer membrane where they are likely responsible for the generation of metabolic peptides required for survival of the pathogen. Both autoprocessing and activity were dependent on calcium concentrations >1 mm, consistent with the protein's activity within the intestinal and inflammatory milieus. Calcium also stabilized the Tpr structure and rendered the protein fully resistant to proteolytic degradation by gingipains. Together, our findings suggest that Tpr is an example of a bacterial calpain, a calcium-responsive peptidase that may generate substrates required for the peptide-fermenting metabolism of P. gingivalis. Aside from nutrient generation, Tpr may also be involved in evasion of host immune response through degradation of the antimicrobial peptide LL-37 and complement proteins C3, C4, and C5. Taken together, these results indicate that Tpr likely represents an important pathogenesis factor for P. gingivalis.

Keywords: calcium; calcium-dependent activity; calpain; enzyme processing; periodontal disease; virulence factor.

Figures

FIGURE 1.
FIGURE 1.
Expression of WT and catalytic mutant Tpr and assessment of their autoprocessing activities. A, both WT (Tpr) and the active-site cysteine-to-serine mutant (TprC229S) were expressed in E. coli and purified. Lane 1, molecular mass marker (kDa); lanes 2 and 3, cell extracts of E. coli before and 3 h after induction of GST-Tpr expression with isopropyl-1-thio-β-d-galactopyranoside, respectively; lane 4, a fusion protein GST-Tpr (84 kDa) obtained from E. coli extract by affinity chromatography on glutathione-Sepharose; lane 5, Tpr55, Tpr48, and Tpr37 after GST was cleaved off by PreScission Protease and removed on glutathione-Sepharose; lane 6, Tpr55, Tpr48, and Tpr37 purified by gel filtration; lane 7, TprC229S (55 kDa) expressed and purified in the same way as WT Tpr. N-terminal sequences of Trp55, TprC229S, Tpr48, and Tpr37, determined by Edman degradation, are shown on the right side. B, the primary structure of Tpr with identified proteolytic cleavage sites (arrows) that generated the Tpr48 (Gly-62–Met-63), Tpr37 (Lys-160–Ala-161 and Asp-166–Ile-167), and Tpr33 (Arg-195–Asp-196) forms of the peptidase. Catalytic residues (Cys-229, His-406, and Asn-426), identified by alignment of the Tpr sequence with papain- and calpain-like peptidases, are in bold font. A domain similar to papain is indicated by a thin underline, starting from Leu-189. Of note, Tpr is encoded without a signal peptide. A pentapeptide (small font) preceding Met-1 is derived from the plasmid. C, Tpr55 and Tpr48 were localized in P. gingivalis W83 outer membrane and cellular extract. Lane 1, whole cellular extract; lane 2, cytoplasmic/periplasmic fraction; lane 3, inner membrane fraction; lane 4, sarkosyl-insoluble outer membrane fraction. Western blot analysis of cell fractions of P. gingivalis were probed with Tpr-specific rabbit antiserum.
FIGURE 2.
FIGURE 2.
Tpr55 is a zymogen requiring Ca2+ for sequential autoproteolytic processing to generate active forms of the enzyme: first Tpr48, then Tpr37, and finally stable Tpr33. A, purified Tpr55/Tpr48 (in buffer containing EDTA) was incubated in assay buffer without calcium (EDTA +/Ca2+ −) or with CaCl2 (EDTA −/Ca2+ +), and samples were treated with DCG-04. After a 15-min incubation at 37 °C, the reaction was terminated by boiling in denaturing sample buffer. Proteins were resolved by SDS-PAGE and subjected to Western blot analysis. Lanes 1 and 2, protein staining; lanes 3 and 4, detection of probe labeled active Tpr. B and C, purified Tpr55/Tpr48 was supplemented with CaCl2 and treated with DCG-04 (at time 0 or after incubation at 37 °C for specific time intervals). A sample incubated in buffer with EDTA (Tpr-ctrl) served as a control. Proteins were resolved by SDS-PAGE, and gels were stained for protein (B) or subjected to Western blot analysis to detect probe labeled proteins (C). SDS-PAGE-separated proteins were subjected to N-terminal sequencing analysis. D, purified Tpr55/Tpr48 was incubated in the assay buffer with Ca2+ at 21 °C for the indicated periods of time, and samples were analyzed by SDS-PAGE.
FIGURE 3.
FIGURE 3.
Tpr hydrolyzes Suc-Leu-Leu-Val-Tyr-AMC and Z-Phe-Arg-AMC with a lag-phase (A), which is apparently due to the enzyme inhibition by the N-terminal remaining non-covalently associated with Tpr33 (B and C). Recombinant Tpr (Tpr55/Tpr48) was preincubated in the assay buffer for 1 h before the substrate was added, and then fluorescence was recorded (λex = 355 nm and λem = 460 nm) for 3 h (A). Tpr processing was activated by calcium (2.5 mm final concentration), and at specific time points aliquots were withdrawn and subjected to the SDS-PAGE analysis (B). Simultaneously, the activity was measured employing Suc-Leu-Leu-Val-Tyr-AMC as the substrate (C). The inset shows substrates hydrolysis by Tpr preincubated in the presence of 2.5 mm CaCl2 for 4 h. RFU, relative fluorescence units.
FIGURE 4.
FIGURE 4.
Tpr hydrolyzes Suc-Leu-Leu-Val-Tyr-AMC at a pH in the range from 6.5 to 9.0 (A), obeying Michaelis-Menten kinetics (B), and the enzyme has a broad specificity (C). The pH profile was determined by assaying the activity in buffers of different pH (all containing 5 mm CaCl2 and 1 mm DTT), with substrate added after 30 min of preincubation. Activity (relative fluorescence units/min at the linear phase of fluorescence increase) at 50 mm Tris-HCl, 5 mm CaCl2, and 1 mm DTT, pH 7.5, was arbitrarily taken as 100% (A). Km values for Suc-Leu-Leu-Val-Tyr-AMC and Z-Phe-Arg-AMC were determined from the initial velocity of hydrolysis of substrates at concentrations in the range from 0.5 μm to 30 μm (B). A Tpr cleavage consensus sequence motif was derived from digestion of bovine casein and human fibrinogen by Tpr33 (C). Residues at positions 6 and 7 are equivalent to P1-P1′. The motif was discovered using MEME (Multiple Em for Motif Elicitation) with default parameters except for site distribution = 0 or 1 set for the presence or absence, respectively, of a given residue.
FIGURE 5.
FIGURE 5.
Tpr33 degrades the fibrinogen (A), fibronectin (B), and LL-37 (C). Fibrinogen (100 μg), fibronectin (50 μg), and LL-37 (10 μg) were incubated at 37 °C in 100 μl of 100 mm Tris-HCl, 5 mm CaCl2, pH 7.5. At the indicated time points, aliquots (10 μl) were withdrawn from the reaction mixture, mixed with 10 μl of reducing SDS-PAGE sample buffer, and denatured at 95 °C for 5 min to stop the reaction. Samples (20 μl) were subjected to SDS-PAGE. Substrates incubated with Tpr33 inhibited with E-64 and TprC229S served as non-digested protein control.
FIGURE 6.
FIGURE 6.
Tpr33 hydrolyzes the complement proteins C3 (A), C4 (B), and C5 (C). Complement compounds (100 μg) were incubated at 37 °C in 100 μl of 100 mm Tris-HCl, 5 mm CaCl2, pH 7.5, up to 12 h at a 5:1 substrate/protease molar ratio. At the indicated time points, aliquots (10 μl) were withdrawn from the reaction mixture, mixed with 10 μl of reducing SDS-PAGE sample buffer, and denatured at 95 °C for 5 min to stop the reaction. Samples (20 μl) were subjected to SDS-PAGE. Substrates incubated with Tpr33 inhibited with E-64 and TprC229S served as non-digested protein control.
FIGURE 7.
FIGURE 7.
TprC229S is impervious to proteolysis by active forms of Tpr (A) and in the presence of Ca2+ also blocks degradation by Kgp (B) and Rgp (C) because the protein is stabilized (D). Recombinant TprC229S was incubated with Tpr55/Tpr48 at 37 °C in the activity assay buffer at the molar ratio 100:1 for the indicated time periods (A) or with gingipains at different molar ratios in the presence or absence of Ca2+ for 1 h (B and C). Boiling in a denaturing sample buffer stopped the reaction, and samples were analyzed by SDS-PAGE followed by protein staining and analysis by N-terminal sequencing. The effect of increasing concentrations of calcium ions on the Tm of TprC229S was measured by differential scanning fluorimetry (D).

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