Amidase

The genes for the full-length LysGH15 and its three individual domains (i.e., CHAP, amidase-2, and SH3b) were amplified using corresponding primers that were designed based on the full-length lysGH15 gene (GenBank: AY176327) and were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The coding regions for the CHAP (residues 1–165), amidase-2 (residues 165–403), and SH3b (residues 368–495) domains were cloned into the pMCSG7 vector as previously reported [41] (link). The full-length lysGH15 gene was subcloned into the pET-26b vector. Mutations were designed based on these constructs and were generated using the QuikChange Site-Directed Mutagenesis Kit following the manufacturer’s instructions (Stratagene). All of the recombinant plasmids were sequenced to verify the sequence.
The plasmids harboring the target gene, which encoded 6× His-tagged proteins, were transformed into E. coli BL21(DE3) (Tiangen Biotechnology). The cells were grown in Luria-Bertani (LB) medium at 37°C until the OD₆₀₀ reached 0.8. The culture was then induced with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) for 20 h at 16°C. Cells were harvested by centrifugation at 4,670×g for 30 min and were resuspended in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 50 mM Na₂HPO₄, and 10 mM KH₂PO₄, pH 7.4). After lysis by sonication, the cell debris was removed by centrifugation at 38,900×g for 30 min. The supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA) resin gravity column (Qiagen) that had been previously equilibrated with PBS. The column was washed using 100 ml of lysis buffer containing 20 mM imidazole, followed by a 50 mM imidazole wash. Finally, the protein was eluted with PBS containing 500 mM imidazole. After buffer exchange, the 6× His-tag was removed using tobacco etch virus (TEV) proteolysis (except for full-length LysGH15). Uncut protein was removed using a second Ni-affinity chromatography step. The proteins without a His-tag were concentrated and applied to a Superdex G200 size-exclusion chromatography column (Amersham) that was preequilibrated with 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl (500 mM NaCl for full-length LysGH15). For the SH3b domain, 40 mM Na₃PO₄ and 50 mM NaCl, pH 6.5, were used. Fractions containing the purified target protein were pooled and stored at −80°C until further analysis.
The E. coli BL21(DE3) strain that contained the pMCSG7-CHAP vector was grown in M9 medium containing glucose (0.2% M/V), MgSO₄ (1 mM), and ampicillin (100 µg/ml) at 37°C until the OD₆₀₀ reached 0.8. Subsequently, selenomethionine was added to the culture (50 µg/ml). The subsequent purification steps were similar to those used for the native protein.
The plasmid pMCSG7-SH3b was transformed into E. coli BL21(DE3). The cells were grown in M9 medium containing glucose (0.2% M/V), MgSO₄ (1 mM), and ampicillin (100 µg/ml). ¹⁵N ammonium chloride and/or ¹³C glucose was used as the sole nitrogen and carbon sources, respectively, for isotope labeling. Labeled SH3b was purified using an identical procedure as that used for the native protein.

Plasma samples were prepared from citrate-blood collections, centrifuged to prepare the platelet-free plasma and immediately frozen at −80°C. The spontaneous amidase activity was evaluated using the peptide substrate HD-Pro-Phe-Arg-pNA (1 mM; Bachem), representing the P1-P′1 scissile bond by kallikrein at the C-terminus of BK. This assay refers to enzymes with spontaneous amidase activity, i.e. the Serine proteases of contact phase and fibrinolysis (kallikrein, FXII, plasmin and tissue-type plasminogen activator). Spontaneous amidase activity was kinetically monitored by the A₄₀₅ at 30°C (ThermoFisher Spectrophotometer), and expressed in nmol⋅min⁻¹⋅mL⁻¹ (molar extinction coefficient of p-nitroaniline 8800 M⁻¹⋅cm⁻¹).
In order to refer to plasma proenzyme activation, the contact system was activated by cold pre-incubation of plasma sample with dextran sulfate (12.5 mg⋅mL⁻¹) [18] (link), then the subsequent enzyme activity was assessed using the peptide substrate HD-Pro-Phe-Arg-pNA and monitored by the A₄₀₅ at 30°C.

Cells were plated in 96-well plates at 20,000 cells per well with 25 μl in the appropriate medium (see above). SACLAC, SOCLAC, and DMSO were prepared in medium supplemented with 20% FBS at the appropriate concentrations from a DMSO stock solution, and 25 μl were dispensed onto the 96-well plate. Cells were incubated with the indicated concentrations of SACLAC, URB597, or DMSO (vehicle control) for 1 h or the indicated times in a humidified incubator at 37°C and 5% CO₂. For time-course studies, cells were resuspended in inhibitor-free culture medium before RBM1-151 incubation. Unless indicated otherwise, after incubation with the inhibitors, 50 μl of RBM1-151 was dispensed onto the cells at a final concentration of 20 μM in medium with 20% FBS. Cells were incubated with RBM1-151 for 1 h in a humidified incubator at 37°C and 5% CO₂. After RBM1-151 incubation, 25 μl of 100% methanol was added to each well. Immediately after, 100 μl of 2.5 mg/ml sodium periodate in 100 mM glycine (pH 10.6) was added to each well. The plate was incubated in a humidified incubator at 37°C and 5% CO₂ for 30 min. Fluorescence was measured at 355 nm excitation and 460 nm emission using a microtiter plate reader. During data analysis, background signal from RBM1-151 in culture media without cells was subtracted from all values, and each amidase activity was calculated using the following equation: [umbelliferone]_C - [umbelliferone]_I, where [umbelliferone] corresponds to the amounts in μM/h/2 × 10⁴ cells produced in both control cells (C, treated with DMSO) and cells treated with each inhibitor (I) calculated from an umbelliferone calibration curve.

The RM contained 5 mM αKGM, 5 mM DTT, 100 mM Tris-HCl buffer (pH 8.5), and an enzyme source. The final RM volume was adjusted with H₂O to 50 µL. Note that when the assays were conducted with purified ωA, the blank contained complete RM lacking enzyme. For assays of crude tissue/cell homogenates, the blank contained the complete RM plus homogenate and 200 mM glycylglycine. After 5–30 min incubation at 37 °C, the reaction was terminated by the addition of 20 µL of 5 mM 2,4-dinitrophenylhydrazine in 2 M HCl. After a further incubation for 5 min at 37 °C, 130 µL of 1 M NaOH was added, and the absorbance was read at 430 nm within 5 min. The ε_430nm of α-ketoglutarate*2,4-dinitrophenylhydrazone under these conditions was 16,000 M⁻¹·cm⁻¹.