PI fractionation

Intact potato PI were separated by anion-exchange chromatography, according to the method as described [6]. In short, a commercial PI-enriched protein concentrate was diluted to a final concentration of 6% and the pH was adjusted to pH 8.0 with 1 M NaOH. After centrifugation (10 min, 4500 g) to remove insoluble protein and the clear supernatant was kept as is (fraction 1) or was subjected to anion-exchange chromatography on an Äkta Pure FPLC system equipped with an XK16 column containing Q-Sepharose Fastflow resin (Column volume 24 mL). Equilibration and sample application were done at pH 8, and proteins were eluted with a linear NaCl gradient (0–250 mM). Three peaks were obtained and collected in fractions of 5 mL, which were pooled and freeze dried. The freeze-dried material was redissolved in MilliQ and desalted on 3-kDa MWCO spin filters (Macrosep® Centrifugal Filters, Pall), using diafiltration with MilliQ water (fractions 2 and 3).

Protein hydrolysis

Heat-coagulated protein was hydrolyzed using two different commercial proteases (fractions 4 and 5): Esperase® 8.0 L or Protamex® (Novozymes A/S, Bagsvaerd, Denmark). Incubations were performed in batch setup using 10% w/v substrate, at 60 °C in 0.1 M Tris–HCl buffer at pH 8.0, with continuous stirring with a top stirrer (150 rpm) and without pH control. The concentration of the enzymes was 0.5% (w/v) in both cases. After 2.5 h, the proteases were inactivated by heat incubation (15 min at 90 °C) and the liquid and solid fractions were separated by centrifugation (4000 g, 15 min). The liquid phase containing the hydrolysates was decanted, filtered on Whatman paper (Grade 595) to remove residual solids, and freeze-dried. Samples were re-dissolved in MilliQ and desalted by diafiltration over 1-kDa MWCO spin filters (Macrosep® Centrifugal Filters), according to supplier instructions. Protein concentrations were determined by the use of a Kjeldahl-calibrated (Nx6.25) Sprint Protein Analyzer (CEM Corporation, Matthews). Lipopolysaccharide was removed from the fractions using ε-poly-l-lysine-agarose (Pierce High-Capacity Endotoxin Removal Spin Column, 1 mL, #88,276; Thermo Fisher Scientific, Inc.), in accordance with the manufacturer’s instructions.

Inhibition of ACE1, ACE2, cathepsin B, cathepsin L, collagenase, and furin

ACE2, cathepsin B, cathepsin L, and furin protease activities were measured with the following fluorometric kits: ACE2 Inhibitor Screening Kit (#K310 BioVision), Cathepsin L Inhibitor Screening Kit (#K161-100, BioVision), Cathepsin B Inhibitor screening kit (MAK200, Sigma-Aldrich), and Furin Protease Assay Kit (#78,040 BPS Bioscience). Shortly, potato PI fractions were diluted in phosphate-buffered saline (PBS) and added to a 96-well black well plate with four positive, two negative, and two buffer controls per plate. Activities were measured on a fluorescence plate reader (Synergy H1) at the appropriate ex/em wavelength with automatic gain adjustment over a 1-h period at 1-min intervals. The resulting enzyme activities were reported as the maximum increase in fluorescence intensity over time per well. Inhibitory activities were calculated as percentages of intensity increase over time that were lost relative to the average of the positive controls. For the different preparations, various concentrations were tested to calculate the IC50 value by linear regression of the residual activity against log(concentration). ACE1 and collagenase protease activities were measured with following colorimetric kits: ACE1 Inhibitor Screening Kit (K719-100 from BioVision obtained from Bio-Connect, The Netherlands) and Collagenase Activity Colorimetric Kit (MAK293 Sigma-Aldrich).

Inhibition of elastase

Inhibition of elastase activity was determined according to two methods. First, elastase inhibition activity was measured according to Valueva et al. [49] with minor modifications. A substrate solution was prepared by dissolving 10 mg of N-succinyl-ala-ala-ala-p-nitroanilide (Sigma-Aldrich, S4760-100 mg) in 50 µL DMSO. A solution of 15 mL of a 50-mM Tris buffer (Merck 1.08382.1000) at pH 8.2 and 5 mM CaCl2 (Sigma-Aldrich, C3881) was preheated to 50 °C and added to the substrate solution. Elastase concentrate human neutrophil elastase (50 µL; Calbiochem, 324,681) was diluted in 10 mL of 1 mM HCl/5 mM CaCl2 solution to form the enzyme solution. Sample solutions of potato protein were diluted in demineralized water to such an extent that inhibitory activities between 20 and 80% were obtained. Activities were measured by introducing 25 µL of enzyme solution and 25 µL of sample into the wells of a 96-well microtiter plate. For sample blanks, demi water was added instead of enzyme. For positive controls, demi water was added instead of sample. For negative controls, 25 µL of acetic acid was added instead of sample. The reaction was started by adding 125 µL of substrate solution to the wells. Hydrolysis of the substrate by the enzyme results in the release of p-nitroanilide with a Lambda-max of 405 nm. This was measured for 300 s at 20-s intervals on a MultiSkan Go (ThermoScientific). Activities were determined by linear regression of the A405 over time. Second, elastase inhibition activity was measured with fluorogenic kit (#E12056, Invitrogen). Shortly, pig pancreas elastase (Invitrogen, cat. no. E12056) at a concentration of 1 μg/mL was incubated with different concentrations of extract (1.000–0.5 μg/mL) or a known mixture of protease inhibitors (Halt protease inhibitor mixture, # 78437, Thermofisher) and the remaining proteolytic activity was determined by measuring the catalysis of fluorogenic elastin (15 μg/mL DQ-elastin, #E12056, Invitrogen).

Inhibition of trypsin and chymotrypsin

Trypsin inhibitory activity and chymotrypsin inhibitory activity were measured essentially as previously described [50]. Briefly, chromogenic substrates (l-BAPNA, Na-benzoyl-l-arginine-p-nitroanilide from Sigma-Aldrich (B4875), and l-PAPNA, N-succinyl-l-phenylalanine-p-nitroanilide from Sigma-Aldrich (S4760)) were hydrolyzed by either trypsin or chymotrypsin in 50 mM Tris/HCl buffer (GE PlusOne Tris) of pH 8.5 in the presence of 5 mM CaCl2 (Sigma-Aldrich C1016) in 96-well plates. This resulted in an increase in absorbance at 405 nm, which was monitored on a ThermoScientific Multiskan GO plate reader. Increasing quantities of inhibitor were added simultaneously to wells that were otherwise identical, in order to reduce the protease signal. The amount of protease that was rendered inactive per amount of inhibitor was then calculated by linear regression.

Inhibition of papain

Papain inhibitory activity was performed according to the method of Pouvreau [20]. Briefly, l-BAPNA was exposed to papain (Applichem A3824) in a 100-mM potassium phosphate buffer of pH 6.5 (Merck 1.05099) containing 300 mM KCl (VWR Rectapur 26759.291), 4 mM EDTA (Sigma-Aldrich 27285), and 16 mM of cysteine (Fluka 30130). The hydrolysis of the substrate was monitored at 405 nm in a 96-well plate with different wells containing increasing quantities of inhibitor. The amount of protease inhibited per amount of sample material was obtained by linear regression.

Inhibition of MMP-9

Recombinant human proMMP-9 was produced in Sf-9 insect cells and activated by incubation with the catalytic domain of MMP-3 as previously described [51]. Activation of proMMP-9 was confirmed by a band shift of ± 10 kDa on SDS-PAGE, corresponding to the removal of the auto-inhibitory propeptide domain. To test the inhibitory activity of the potato extracts, we measured the degradation of a fluorogenic MMP substrate peptide (2.5 μg/mL OmniMMP, Mca-PLGL-Dpa-AR-NH2, cat. no. BML-P126-0001, Enzo Life Sciences, Farmingdale, NY) by MMP-9 (0.1 nM) in the presence or absence of different concentrations of extract (1000–0.5 μg/mL). As a negative control, we included the known MMP-9 inhibitor SB-3CT (dose-response) and EDTA (100 mM).

Inhibition of proteases in human neutrophil degranulates

Neutrophils were isolated from fresh blood of healthy donors, via density gradient centrifugation as described [52]. To obtain neutrophil degranulate, neutrophils were suspended in degranulation buffer (120 mM NaCl, 15 mM CaCl2, 20 mM Tris/HCl pH 7.5) at a concentration of 107 cells/mL and degranulation was induced by stimulation with N-formylmethionyl-leucyl-phenylalanine (fMLF) (final concentration 0.5 µM) for 20 min at 37 °C. Next, the supernatant was collected by centrifugation. Proteolysis of an MMP substrate (OmniMMP, broad substrate for most MMP and for ADAM17/TACE) and DQ-elastin by proteases in the neutrophil degranulate (10 µL) was measured in the presence of absence of different concentrations of fractions 1–5, EDTA (a metalloprotease inhibitor), and AEBSF (a serine protease inhibitor).

Vero E6 cell line for virus infection assays

The African green monkey Vero E6 cell line (ATCC CRL-1586) was maintained in Dulbecco’s minimal essential medium (DMEM) (Gibco), high glucose supplemented with 10% fetal bovine serum (Life Science Production), penicillin (100 U/mL), and streptomycin (100 U/mL) (Gibco). Vero E6 cells were mycoplasma negative and maintained at 37 °C under 5% CO2.

SARS-CoV-2 production and characterization

SARS-CoV-2 was isolated from an infected patient via a throat swab. Sequence analysis revealed that the isolated strain is an alpha SARS-CoV-2 variant and is mycoplasm-negative. The original stock was passaged twice in Vero E6 cells to obtain a working stock. Infectious virus titers were determined by plaque assay on Vero E6 cells and defined as the number of plaque forming units (PFU) per mL. The detection limit of the assay is 150 FU/mL [25].

Cytotoxicity assay

Vero E6 cells were seeded into 96-well plates at a density of 2 × 104 cells per well. The following day, cells were exposed to increasing concentrations of PI fractions for 19 h at 37 °C under 5% CO2. PI fractions were diluted in cell culture medium. Subsequently, cellular cytotoxicity was evaluated using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit using manufacturer’s instructions from Promega (Madison, WI). Briefly, at 19 h post-treatment, 20 µL of MTS/PMS solution was added per well and incubated for 2 h at 37 °C. Subsequently, 10% SDS was added to each well (2% end concentration) to stop the reaction and the absorbance was measured at 490 nm with a microplate reader.

Antiviral assay in Vero E6 cells

Vero E6 cells were seeded at a density of 1.3 × 105 cells/well into 12-well plates. The following day, cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) 1 in presence of increasing concentrations of PI. Infection was done in 250 µL DMEM (2% FBS) medium. At 2 hpi, the virus inoculum was removed, cells were washed twice with plain DMEM medium and fresh DMEM with 10% FBS, and PI were added after which incubation was continued for 6 h. Finally, the cell supernatant was collected and centrifuged to clear from cell debris and the viral titer was determined using plaque assay. Resveratrol was used as positive control in reducing virus transduction; its vehicle (Ethanol) was an additional control.

Vero E6 cell based ACE2 enzymatic activity assay

Vero E6 cells were plated at a density of 10 × 105 cells into a 96-well plate and incubated overnight at 37 °C, in a humidified 5% CO2 atmosphere in DMEM (Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS) (Sigma Aldrich, F7524, St. Louis, MO, USA). Concentrations between 100 nM and 1 μM were used for fractions 1–5 and incubated for 6 h at 37 °C, whereafter 2 µL of the ACE2 protease substrate solution (Abcam, UK #111,750) was added. Ex/Em was 485/530 nm for the measurement of the proteolysis.

Proteomics analysis

Fractions 1, 2, 3, and 4 were selected for proteomics analysis. Protein samples were loaded on an 8% pre-cast RunBlue gel (Expedeon) and run at 100 V for 5 min. Gel staining was performed using InstantBlue (Expedeon) followed by a wash with ultrapure water. Coomassie-stained bands were excised in one gel slice that was further cut into small pieces and destained using 70% 50 mM NH4HCO3 and 30% acetonitrile. Reduction was performed using 10 mM DTT dissolved in 50 mM NH4HCO3 for 30 min at 55 °C. Next, the samples were alkylated using 55 mM chloroacetamide in 50 mM NH4HCO3 for 30 min at room temperature and protected from light. Subsequently, samples were washed for 10 min with 50 mM NH4HCO3 and for 15 min with 100% acetonitrile. Remaining fluid was removed, and gel pieces were dried for 15 min at 55 °C. Tryptic digestion was performed by addition of sequencing-grade modified trypsin (Promega; 25 µL of 10 ng/mL in 50 mM NH4HCO3) and overnight incubation at 37 °C. Peptides were extracted using 5% formic acid in water followed by a second elution with 5% formic acid in 75% acetonitrile. Samples were dried in a SpeedVac centrifuge and dissolved in 20 µL 5% formic acid in water for analysis with LC-MS/MS.

The samples were analyzed on a nanoLC-MS/MS consisting of an Ultimate 3000 LC system (Dionex, Amsterdam, The Netherlands) interfaced with a Q-Exactive plus mass spectrometer (Thermo Fisher Scientific). Peptide mixtures were loaded onto a 5 mm × 300 μm i.d. C18 PepMAP100 trapping column with water with 0.1% formic acid at 20 μL/min. After loading and washing for 3 min, peptides were eluted onto a 15-cm × 75-μm i.d. C18 PepMAP100 nanocolumn (Dionex). A mobile phase gradient at a flow rate of 300 nL/min and with a total run time 75 min was used: 2–30% of solvent B in 87 min; 30–80% B in 5 min; 90% B during 1 min, and back to 2% B in 0.1 min. Solvent A was 100:0 water/acetonitrile (v/v) with 0.1% formic acid, and solvent B was 0:100 water/acetonitrile (v/v) with 0.1% formic acid. In the nanospray source, a stainless-steel emitter (Thermo Fisher Scientific) was used at a spray voltage of 2 kV with no sheath or auxiliary gas flow. The ion transfer tube temperature was 250 °C. Spectra were acquired in data-dependent mode with a survey scan at m/z 300–1650 at a resolution of 70.000 followed by MS/MS fragmentation of the top 10 precursor ions at a resolution of 17,500. Singly charged ions were excluded from MS/MS experiments, and fragmented precursor ions were dynamically excluded for 20 s.

The PEAKS Studio version Xpro (Bioinformatics Solutions, Inc., Waterloo, Canada) software was used to search the MS data against a protein sequence database of potato Solanum tuberosum [53] derived from the work of Xu et al. [54]. Search parameters include trypsin digestion with up to two missed cleavages, fixed modification carbamidomethylation of cysteine, variable modification oxidation of methionine, precursor mass tolerance of 20 ppm, and fragment mass tolerance of 0.02 Da. The false discovery rate was set at 0.1% on the peptide level.