Journal: Journal of Biological Chemistry

Article Title: Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

doi: 10.1074/jbc.m114.601716

Figure Lengend Snippet: FIGURE 1. T. forsythia open reading frame ID TF0781 (Oralgen) encodes an inhibitory active serpin. A, multisequence alignment of the inhibitory core of T. forsythia serpin with squamous cell carcinoma antigen 1, SCCA1 (P29508), human 1-antitrypsin (UniProt accession number P01009) and B. longum serpin (UniProtaccessionnumberQ8G7X7).ThepositionsP1-P1areframed.B,possibletranslationinitiationsitesoftheserpinfromT. forsythia.TheT. forsythiaserpin locus TF0781 was resequenced together with the 5 and 3 flanking regions, and putative sites of initiation of translation (Tfs62, TFs55, and TFs46) according to a non-classical mechanism operating in the Bacteroidetes phylum were determined. The underline sequence in TFs46 was predicted using LipoP 1.0 Server to represent a signal peptide typical for lipoproteins. C, neutrophil elastase (NE) was preincubated with increasing amounts of T. forsythia cells suspension. The residual enzyme activity was determined and plotted against the volume of suspension (average of two independent assays). The inset shows NE inhibition by washed T. forsythia cells (WC), bacterial cells homogenate (CH), soluble cytoplasm/periplasm proteins (C/P), and cell envelope (CE) subcellular fractions standardized to the same volume of the initial culture. The mean value from two independent experiments is presented. D, three putative variants of serpin were cloned into the pGex-6P-1 vector, expressed as fusion proteins with GST in E. coli, and purified by affinity chromatography on glutathione-Sepharose. The expressed proteins were analyzed by SDS-PAGE. E, inhibitory activity of the recombinant proteins was determined using neutrophil elastase as the target protease.

Article Snippet: Enzymes, Inhibitors, Substrates, and Other Reagents—The following enzymes and reagents were used: human neutrophil elastase, cathepsin G, ecotin, and human 2-macroglobulin (BioCentrum, Krakow, Poland; Athens Research and Technology, Athens, GA); porcine pancreatic elastase (Promega, Fitchburg, WI); bovine pancreatic -chymotrypsin, bovine pancreatic trypsin, thrombin from human plasma, subtilisin Carlsberg, benzoyl-Arg-pNA,2 Boc-Val-Pro-Arg-aminofluoromethylcoumarin, Boc-Gln-Ala-Arg-7-amino-4-methylcoumarin (AMC), and metoxysuccinyl-Ala-Ala-Pro-Val-pNA (Sigma); succinyl-Ala-Ala-Pro-Phe-pNA and 7-methoxycoumarin-4-acetic acid (MCA)-FVT-4-guanidine-phenylalanine (Gnf)-SW-Anb-NH2 (Anb is the amide of amino benzoic acid; Merck).

Techniques: Sequencing, Suspension, Activity Assay, Inhibition, Clone Assay, Plasmid Preparation, Purification, Affinity Chromatography, SDS Page, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

doi: 10.1074/jbc.m114.601716

Figure Lengend Snippet: FIGURE 2. Expression and purification of recombinant miropin. A, SDS- PAGE of E. coli extracts and tag-free miropin after purification on glutathione- Sepharose and in-column digestion with the PreScission protease. Lane 1, E. coli extracts before the addition of isopropyl-1-thio--D-galactopyrano- side; lane 2, E. coli extracts at 6 h after isopropyl-1-thio--D-galactopyrano- side-induced protein expression; lane 3, tag-free miropin (46.1 kDa). B, profile of gel filtration chromatography of miropin on HiLoad 16/60 Superdex 75 pg column. mAU, milliabsorbance units. C, SDS-PAGE of two miropin fractions resolved in B: lane 1, miropin before gel filtration; lane 2, peak 1; lane 3, peak 2. The presence of miropin in the major band was confirmed by N-terminal sequence analysis. D, inhibitory activity against human neutrophil elastase (NE) (0.1 g) determined in the different gel filtration fractions.

Article Snippet: Enzymes, Inhibitors, Substrates, and Other Reagents—The following enzymes and reagents were used: human neutrophil elastase, cathepsin G, ecotin, and human 2-macroglobulin (BioCentrum, Krakow, Poland; Athens Research and Technology, Athens, GA); porcine pancreatic elastase (Promega, Fitchburg, WI); bovine pancreatic -chymotrypsin, bovine pancreatic trypsin, thrombin from human plasma, subtilisin Carlsberg, benzoyl-Arg-pNA,2 Boc-Val-Pro-Arg-aminofluoromethylcoumarin, Boc-Gln-Ala-Arg-7-amino-4-methylcoumarin (AMC), and metoxysuccinyl-Ala-Ala-Pro-Val-pNA (Sigma); succinyl-Ala-Ala-Pro-Phe-pNA and 7-methoxycoumarin-4-acetic acid (MCA)-FVT-4-guanidine-phenylalanine (Gnf)-SW-Anb-NH2 (Anb is the amide of amino benzoic acid; Merck).

Techniques: Expressing, Purification, Recombinant, SDS Page, Filtration, Chromatography, Sequencing, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

doi: 10.1074/jbc.m114.601716

Figure Lengend Snippet: FIGURE 3. Determination of the SI of serine proteases by miropin. A, cathepsin G. B, human neutrophil elastase. C, porcine pancreatic elastase. D, subtilisin Carlsberg. E, trypsin. Each protease was preincubated with increasing concentrations of miropin for 15 min, and residual enzymatic activity was determined and plotted against the serpin:enzyme molar ratio (I0/E0). Activity of enzymes in the absence of inhibitor was considered as 100%. The results presented are the mean S.D. from three experiments.

Article Snippet: Enzymes, Inhibitors, Substrates, and Other Reagents—The following enzymes and reagents were used: human neutrophil elastase, cathepsin G, ecotin, and human 2-macroglobulin (BioCentrum, Krakow, Poland; Athens Research and Technology, Athens, GA); porcine pancreatic elastase (Promega, Fitchburg, WI); bovine pancreatic -chymotrypsin, bovine pancreatic trypsin, thrombin from human plasma, subtilisin Carlsberg, benzoyl-Arg-pNA,2 Boc-Val-Pro-Arg-aminofluoromethylcoumarin, Boc-Gln-Ala-Arg-7-amino-4-methylcoumarin (AMC), and metoxysuccinyl-Ala-Ala-Pro-Val-pNA (Sigma); succinyl-Ala-Ala-Pro-Phe-pNA and 7-methoxycoumarin-4-acetic acid (MCA)-FVT-4-guanidine-phenylalanine (Gnf)-SW-Anb-NH2 (Anb is the amide of amino benzoic acid; Merck).

Techniques: Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

doi: 10.1074/jbc.m114.601716

Figure Lengend Snippet: FIGURE 4. Progress curve analysis of protease inhibition by miropin. A, cathepsin G (1 nM). B, human neutrophil elastase (1 nM). C, porcine pancreatic elastase (1 nM). D, subtilisin Carlsberg (0.05 nM). E, trypsin (0.1 nM). Proteases were added to mixtures containing a constant amount of substrate and increasing concentrations of miropin. Changes in fluorescence (RFU) or absorbance (Abs) were then recorded. The number associated with each progress curve (insets) represents the concentration of miropin (nM). The values of Kobs were plotted as a function of miropin concentration; kass was determined from the slope of the fitted linear curve to the data points and corrected for the stoichiometry factor. With the exception of cathepsin G, the mean S.D. from three experiments is presented.

Article Snippet: Enzymes, Inhibitors, Substrates, and Other Reagents—The following enzymes and reagents were used: human neutrophil elastase, cathepsin G, ecotin, and human 2-macroglobulin (BioCentrum, Krakow, Poland; Athens Research and Technology, Athens, GA); porcine pancreatic elastase (Promega, Fitchburg, WI); bovine pancreatic -chymotrypsin, bovine pancreatic trypsin, thrombin from human plasma, subtilisin Carlsberg, benzoyl-Arg-pNA,2 Boc-Val-Pro-Arg-aminofluoromethylcoumarin, Boc-Gln-Ala-Arg-7-amino-4-methylcoumarin (AMC), and metoxysuccinyl-Ala-Ala-Pro-Val-pNA (Sigma); succinyl-Ala-Ala-Pro-Phe-pNA and 7-methoxycoumarin-4-acetic acid (MCA)-FVT-4-guanidine-phenylalanine (Gnf)-SW-Anb-NH2 (Anb is the amide of amino benzoic acid; Merck).

Techniques: Inhibition, Fluorescence, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

doi: 10.1074/jbc.m114.601716

Figure Lengend Snippet: FIGURE 5. Detection of stable covalent complexes of miropin with serine proteases. A–E, SDS-PAGE of miropin incubated with increasing concentrations of serine proteases at a molar ratio ranging from 0.25–2 as indicated. Little amounts of complexes of miropin with target proteases, especially in case of subtilisin (D) and trypsin (E) result from the variability in the SDS stability of the various complexes and/or different susceptibility of the covalent complexes to hydrolysis by non-inhibited proteases during SDS-PAGE sample preparation. F, effect of NaOH on the stability of the miropin-human neutrophil elastase complex. Cat G, cathepsin G; NE, neutrophil elastase; PE, pancreatic elastase.

Article Snippet: Enzymes, Inhibitors, Substrates, and Other Reagents—The following enzymes and reagents were used: human neutrophil elastase, cathepsin G, ecotin, and human 2-macroglobulin (BioCentrum, Krakow, Poland; Athens Research and Technology, Athens, GA); porcine pancreatic elastase (Promega, Fitchburg, WI); bovine pancreatic -chymotrypsin, bovine pancreatic trypsin, thrombin from human plasma, subtilisin Carlsberg, benzoyl-Arg-pNA,2 Boc-Val-Pro-Arg-aminofluoromethylcoumarin, Boc-Gln-Ala-Arg-7-amino-4-methylcoumarin (AMC), and metoxysuccinyl-Ala-Ala-Pro-Val-pNA (Sigma); succinyl-Ala-Ala-Pro-Phe-pNA and 7-methoxycoumarin-4-acetic acid (MCA)-FVT-4-guanidine-phenylalanine (Gnf)-SW-Anb-NH2 (Anb is the amide of amino benzoic acid; Merck).

Techniques: SDS Page, Incubation, Sample Prep

Journal: Journal of Biological Chemistry

Article Title: Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

doi: 10.1074/jbc.m114.601716

Figure Lengend Snippet: FIGURE 6. Identification of cleavage sites of miropin by target proteases. A, miropin was incubated with serine proteases, and the reaction was stopped by adding reducing sample buffer. Proteins were then resolved by SDS-PAGE and electrotransferred onto a PVDF membrane. The stained protein bands, labeled with frames as shown, were excised and subjected to N-terminal sequencing. B, sequences determined for each protease. Cat G, cathepsin G; NE, neutrophil elastase; PE, pancreatic elastase.

Article Snippet: Enzymes, Inhibitors, Substrates, and Other Reagents—The following enzymes and reagents were used: human neutrophil elastase, cathepsin G, ecotin, and human 2-macroglobulin (BioCentrum, Krakow, Poland; Athens Research and Technology, Athens, GA); porcine pancreatic elastase (Promega, Fitchburg, WI); bovine pancreatic -chymotrypsin, bovine pancreatic trypsin, thrombin from human plasma, subtilisin Carlsberg, benzoyl-Arg-pNA,2 Boc-Val-Pro-Arg-aminofluoromethylcoumarin, Boc-Gln-Ala-Arg-7-amino-4-methylcoumarin (AMC), and metoxysuccinyl-Ala-Ala-Pro-Val-pNA (Sigma); succinyl-Ala-Ala-Pro-Phe-pNA and 7-methoxycoumarin-4-acetic acid (MCA)-FVT-4-guanidine-phenylalanine (Gnf)-SW-Anb-NH2 (Anb is the amide of amino benzoic acid; Merck).

Techniques: Incubation, SDS Page, Membrane, Staining, Labeling, Sequencing