Cathepsin g

As a standard: cathepsin L from the human liver (M = 24.2 kDa), RKLLW-NH_2—selective cathepsin L inhibitor (Arg-Lys-Leu-Leu-Trp-NH₂, M = 405.50 kDa), recombinant matrix metalloproteinase-2 (MMP-2), human albumin, cathepsin D, cathepsin G, 1-octadecanothiol (ODM, all Sigma Steinheim, Germany), recombinant matrix metalloproteinase-1 (MMP-1, Wuhan USCN, Hubei), cathepisn B, cathepsin E, (all Calbiochem, Merck Ltd.) were used. Photopolymer ELPEMER SD 2054, hydrophobic protective paint SD 2368 UV SG-DG (PETERS, Kempen, Germany) were used. Also, HBS-ES buffer pH = 7.4 (0.01 M HEPES, 0.15 M sodium chloride, 0.005% Tween 20, 3 mM EDTA), phthalate buffer pH: 2.20, 3.00, acetate buffer pH: 4.0, 4.5, 4.99, 5.57, Michaelis phosphate buffer pH: 6.52, 7.40 (all BIOMED, Lublin, Poland) were used. The aqueous solutions were prepared with miliQ water (Simplicity^®MILLIPORE). Absolute ethanol, acetic acid, sodium chloride, sodium acetate (all POCh, Gliwice, Poland), high purity (99.999%) argon N 5.0 (AIR LIQUIDE, Poland), and ELISA kit (cat. No SEA306Hu, Cloud—Clone Corp., USA) were used.

The following agents were used: ATP (Sigma Aldrich Co., St. Louis, MO; MP Biomedicals, Solon, OH, USA, Calbiochem Co., La Jolla, CA, and Roche Diagnostic Co., Indianapolis, IN), ADP (Sigma), AMP (Sigma), adenosine (Sigma), benzoylbenzoyl ATP (Sigma), oxidized ATP (Sigma), suramin (Wako, Tokyo, Japan), MIA (methyl isobutyl amiloride, Wako), DIDS (4,4′- Diisothiocyanatostilbene-2,2′-disulfonic acid, Sigma), EDTA (Dojindo, Tokyo, Japan), EGTA (Dojindo), dipyridyl (Sigma), CAS (Chrome Azurol S, Sigma), E. coli S17-1 and pK18mobSacB (kindly provided by Dr. F. Taguchi, Okayama University), Instagene matrix (Bio-Rad, Hercules, CA), KOD-Plus (Toyobo, Osaka, Japan), Wizard SV Gel and PCR cleanup system (Promeg, Madison, WI), Ligation High (Toyobo), Protein Assay Rapid Kit (Wako), [¹⁴C] isoleucine (Moravek Biochemicals, Inc, Brea, CA), [¹⁴C]uracil (Moravek Biochemicals, Inc.), α-defensin-1 (Peptide institute, Inc, Osaka, Japan.), cathepsin G (Sigma), vancomycin (Wako), clarithromycin (Taisho-Toyama Pharmaceutical Co., Tokyo), rifampin (Daiichi Sankyo Co., Tokyo), and ethambutol (Sigma), gatifloxacin (Wako), FLUOS (Sigma), LB medium (Invitrogen, San Diego, CA), M9 medium (prepared by our laboratory), Heart infusion agar (Eiken Chemical Co., Tokyo, Japan), Middlebrook 7H9 medium (Becton Dickinson, Cockeysville, MD), and Middlebrook 7H11 medium (Becton Dickinson).

Human recombinant (rec) neuroserpin, purified human plasma α1-ACT, bovine pancreatic α-chymotrypsin, rec apoE3 and apoE4, purified cathepsin G from human leukocytes, purified thrombin from human plasma, and argotroban, were all obtained from Sigma. VLDL-apoE purified from human plasma was from rpeptide. cathepsin G inhibitor was obtained from Calbiochem. PMSF and protease inhibitor mix were from ROCHE. D6E10 anti apoE antibody raised against apoE amino acids 141–160 was from Cell Signaling.

Wild-type (WT) GAS strain M1T1 5448 (M1 GAS) was originally isolated from a patient with necrotizing fasciitis and toxic shock syndrome (21 (link)). An allelic exchange mutant lacking the scl-1 gene (Δscl) and the plasmid-complemented mutant (Δscl + pscl) was generated in a previous report (19 (link)). Lactococcus lactis NZ9000, Staphylococcus aureus Newman strain (ATCC 25904), and M6 GAS JRS4 (M6 GAS) (22 (link)) were also used throughout the study for comparisons. Bacterial strains were cultivated in Todd-Hewitt broth (THB) at 37°C and grown to log phase for all experiments. Where indicated, bacteria were treated with 50 μU cathepsin G (Sigma), 100 µg/mL lysozyme (Sigma), and/or 4 µM LL-37 peptide (AnaSpec) for 60 min at 37°C.

VWF cleavage activity was further analyzed by mass spectrometry as previously described using a synthetic 73-amino-acid peptide (VWF73) (Kokame et al., 2005 (link), Raife et al., 2009 (link)). GBM samples (all diluted to an equivalent elastase concentration of 10 nM) were incubated with FRETS-VWF73 (2 μM final concentration, PeptaNova GmbH, Sandhausen, Germany) in Bis-Tris 5 mM, CaCl₂ 25 mM, Tween-20 0.005%, pH 6.0 (all from Sigma-Aldrich) for 60 min at 30 °C. Neutrophil elastase (10 nM, Sigma-Aldrich), PR3 (10 nM, Euro Diagnostica, Malmö, Sweden) or cathepsin G (10 nM, Sigma-Aldrich) or rADAMTS13 (10 nM, R&D Systems) were included to indicate the molecular masses expected during cleavage. In certain experiments elastase inhibitor (287 μM), anti-PR3 antibody (100 M surplus), cathepsin G inhibitor (1 mM) or EDTA (40 mM) were pre-incubated with the samples for 60 min at 30 °C. FRETS-VWF73 substrate incubated in Bis-Tris buffer alone served as the negative control. Cleavage products of FRETS-VWF73 substrate were analyzed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MALDI Micro MX, Micromass MS Technologies, Manchester, UK).