Serine Proteinase Inhibitors

Recombinant GAS CcpA was purified to homogeneity from Escherichia coli as previously described (Figure 2A) [30] (link). Recombinant GAS HPr was obtained using the same cloning strategy as previously described for recombinant GAS CcpA (Figure 2A) [30] (link). To obtain functional HPrK/P, the GAS hprK/P gene was cloned from strain MGAS5005 into plasmid pET21a (Novagen) with primers designed such that no His tag was attached to the recombinant HPrK/P protein. An E. coli extract enriched for recombinant GAS HPrK/P was created by growing the BL21-HPrK/P cells in LB/ampicillin with 0.5 mM IPTG to an OD₆₀₀ of 2.0. Cells were centrifuged and washed in 20 mM Tris-HCl pH 7.6 with 3 mM DTT, recentrifuged, and lysed via sonication in a buffer containing 0.2 mM Tris-HCl pH 7.6, 0.03 mM DTT, and 0.5 mM PMSF (a serine protease inhibitor). This lysate is enriched for HPrK/P (Figure 2A).
Phosphorylation of HPr at serine-46 was accomplished by incubating 500 µl of recombinant HPr for 20 mins at 37°C with 599 µl of HPrK/P extract in 5 mM ATP, 10 mM fructose-1,6-bisphosphate, 20 mM Tris-HCl pH 7.5, 1 mM DTT, and 5 mM MgCl₂. To obtain purified HPr-Ser46-P, 100 µL of nickel resin (Qiagen) was added and the mixture was rotated for 1 hr at room temperature. The mixture was washed 4 times with 50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, and 20 mM imidazole, and HPr-Ser-46P was eluted with the same buffer except that the imidazole concentration was increased to 250 mM. The phosphorylation state of HPr was assayed by running the unphosphorylated and phosphorylated proteins on a native glycine gel (pH 10.4) (Figure 2B). Repeated analyses showed that phosphorylation of HPr was stable for at least one week.
To maintain CovR in its soluble form and to work with recombinant CovR protein that lacked a tag, we cloned the covR gene from MGAS5005 into plasmid pTXB1 (New England BioLabs) which resulted in a fusion protein with an intein tag and a chitin binding domain. Recombinant CovR was obtained following the manufacturer's instructions with release of the intein tag using DTT (Figure 2A). CovR was phosphorylated as described [25] (link) with phosphorylation assessed by running unphosphorylated and phosphorylated CovR protein under non-denaturing conditions as described for HPr-Ser46-P (Figure 2C). Repeated assays showed a CovR phosphorlyation half-life of about 90 minutes, which is consistent with previous reports [66] (link). Thus, all experiments with phosphorylated CovR were performed immediately following phosphorylation completion. To remove all phosphorylation reagents, CovR was spun through protein desalting columns (Pierce) into freshly made DNA binding buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 2 mM DTT, and 10 µg/mL of polydI:dC). All protein concentrations were assessed using the Bradford assay (Bio-Rad).

F. hepatica newly excysted juveniles (NEJ) were obtained by excysting metacercariae (Italian and Aberystwyth isolates, Ridgeway Research Ltd), as previously described by Robinson et al. [27 (link)]. F. hepatica NEJ were cultured in RPMI-1640 media (ThermoFisher Scientific) supplemented with 30 mM HEPES (ThermoFisher Scientific), 0.1% glucose and 50 μg/ml gentamycin, at 37°C with 5% CO_2, for up to 24 hr. The culture media containing the excretory/secretory (ES) products was collected after 24 hr, concentrated using Amicon Ultra 3kDa columns (Merck Millipore) and stored at -80°C until use [28 (link)].
For the complement blocking assays, the NEJ were washed five times in Dulbecco’s phosphate-buffered saline (DPBS) (ThermoFisher Scientific) and cultured for further 1 hr in 100% NHS at 37°C, 5% CO₂ (1 NEJ/1 μL). Samples of NHS alone were also incubated under the same conditions to be used as control sera (NHS-control). The cultured sera were recovered from each condition following the incubation and stored at -80°C until use in the complement assays.
Activation of the three complement pathways (Classical, Lectin and Alternative) was measured using the Wieslab Complement System Screen (Svar Life Science AB) in a 96-well plate format. Samples of NHS cultured with F. hepatica NEJ or NHS-control were diluted according the manufacturer’s instructions, in the required kit buffer, and incubated at room temperature (RT) for 15 min. Alternatively, to test the effect of the NEJ ES products, NHS was diluted in the required buffers and, after 15 min incubation, RPMI media or NEJ ES (20 μg) with or without the broad-spectrum cathepsin proteinase inhibitor E-64 (20 μM; Sigma-Aldrich) was added to the samples, which were incubated at RT for a further 25 min. The samples (100 μL) were then added to the wells of the Wieslab plates, incubated at 37°C for 1 hr and the activity of each complement pathway measured according to the manufacturer’s instructions. All the assays were performed in triplicate. The complement activity via each pathway, presented as a percentage, was calculated either relative to the activity within the NHS, NHS-control or within NHS assayed with RPMI only, set as 100% activity.
To test the effect of the recombinant F. hepatica rFhSrp1 and rFhSrp2 serpins on the complement pathways, NHS was diluted as above according to the manufacturer’s instructions, and incubated at RT for 15 min. Following which, rFhSrp1 (1 μM), rFhSrp2 (1 μM), rFhSrp1 and 2 combined (referred to hereon in as FhSrps, 1 μM), serine protease inhibitor Futhan (FUT-175; as a positive control in the recommended concentration 100 μM to inhibit complement pathways, BD-Pharmingen-Bioscience), or 1x PBS (negative control) was added to the NHS samples and incubated at RT for a further 25 min, before adding the samples to the wells of the Wieslab plates. The assays were developed as described above. All the assays were performed in triplicate and the percentage inhibition of each complement pathway was calculated relative to the activity within the control NHS samples with PBS.

FASTA sequences from plant-derived peptides and proteins with immunomodulatory, anti-inflammatory, and anticancer bioactivities, i.e., AMPs, OSIPs, and STPIs, were obtained from the entries collected after a protein source search (filtered by plant organism) with the keywords antimicrobial peptide, oxidative stress induced peptide, and serine-type protease inhibitor in the National Center for Biotechnology Information database (NCBI, https://www.ncbi.nlm.nih.gov/protein, accessed on 1 November 2022). For AMPs, the keywords cycloviolacin O₂ from viola odorata, gamma-thionin from capsicum chinense, kalata B1 from oldenlandia affinis, NaD1 from nicotiana alata, pyrularia from pyrularia pubera, and Varv from viola arvensis were also included because, despite the fact that they are not returned under the keyword antimicrobial peptide, they are classified as AMPs with recognized antimicrobial, immune-regulatory, and anticancer activities [9 (link)]. FASTA protein sequences for AMPs, OSIPs, and STPIs can be found in Supplementary Datafiles S1, S2, and S3, respectively.

In vitro incubation analysis of recombinant proteins involved in the interactions between CLIP2, Spz1, and serpins was performed as follows.
Analysis of CLIP2 and proSpz1: 0.25 μg of rCLIP2 and 1.0 μg of proSpz1 were incubated for 30 min at room temperature, and then 0.25 of μg rCLIP2, 1.0 μg of proSpz1, and the incubation mixture were subjected to SDS-PAGE; and the molecular weight changes of proSpz1 were detected by CBB staining and immunoblot analysis.
Incubation analysis of CLIP2 and serpins: 0.25 μg of rCLIP2 and corresponding serpin were mixed and incubated at room temperature for 5 min according to the molar mass ratio of 1:5, and then an equal mass of rCLIP2, serpins and the incubation mixture were subjected to SDS-PAGE. Immunoblot analysis was performed using antibodies against CLIP2, serpin-1a, serpin-6, and the His-tag to determine whether rCLIP2 forms a covalent complex with serpins. As an irreversible serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), which specifically recognize and sulfonate the active site serine residues that determine the activity of serine proteases, is used to block the binding between rCLIP2 and serpin in partial incubation experiments.