Hydrolysis improves the inhibition efficacy of bovine lactoferrin against infection by SARS-CoV-2 pseudovirus

The entry of SARS-CoV-2 into host cells may involve the spike protein cleavage by cathepsin L (CTSL). Certain food proteins such as lactoferrin (Lf) inhibit CTSL. The current study investigated the impact of hydrolysis (0–180 min) by proteinase K on electrophoretic pattern, secondary structure, cathepsin inhibitory and SARS-CoV-2 pseudovirus infectivity inhibitory of bovine Lf. Gel electrophoresis indicated that hydrolysis cut Lf molecules to half lobes (∼40 kDa) and produced peptides ≤18 kDa. Approximation of the secondary structural features through analysis of the second-derivative amide I band collected by infra-red spectroscopy suggested a correlative–causative relationship between cathepsin inhibition and the content of helix-unordered structures in Lf hydrolysate. The half maximal inhibitory concentration (IC50) of Lf hydrolysed for 90 min (H90) against CTSL was about 100 times smaller than that of the Lf hydrolysed for 0 min (H0). H90 had also double activity against SARS-CoV-2 pseudo-types infectivity compared with H0.

A buffer solution of 100 mM ammonium bicarbonate (NH4HCO3) in deionised water was 131 prepared and pH was adjusted to 7.00 ± 0.02 using 0.5 M NaOH. A 0.2 mM solution of Lf in buffer 132 was prepared and the pH was adjusted to 10 ± 0.02, followed by 5 min of slowly shaking at 20 °C. 133 The solution was kept stored for 24 h at 4 °C to warrant complete hydration. Proteinase K and 134 pefabloc SC solutions were prepared in 100 mM NH4HCO3 buffer (pH 7 ± 0.02) at concentrations 135 of 1 mM and 200 mM, respectively. The Lf solution was incubated with proteinase K at 37 ℃ in a 136 shaking water bath and the enzyme to Lf mass ratio was kept at 1:100 (Sharma et al., 1999). The 137 hydrolysis pattern was observed at incubation times of 0, 30, 60, 90, 120, and 180 min. Immediately 138 after hydrolysis, samples were cooled to room temperature (20 ℃) within an ice bath, and 139 supplemented with pefabloc SC at a pefabloc-to-Lf hydrolysate ratio of 1:50 (for proteinase K 140 inactivation), followed by manual shaking for uniform mixing. The hydrolysates were kept at 20 ℃ 141 for 5-10 min and stored at -20 ℃ until further analysis.

Gel electrophoresis
hydrolysate was performed under non-reducing and reducing conditions. The hydrolysate was 147 diluted threefold to a concentration of 0.07 mM, uniformly mixed with a mixture of SDS-PAGE 148 sample buffer (ratio 2:5, v/v) and treated with either 100 mM ammonium bicarbonate buffer (pH 10 149 ± 0.01, for non-reducing conditions) or dithiothreitol (for reducing conditions), followed by 150 centrifugation for 1 min at 800 × g. The samples were heated in a shaking water bath for 10 min at 151 70 °C and centrifuged for 1 min at 800 × g. Aliquots (20 μL) of samples were loaded on 4-12% bis-

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Tris precast polyacrylamide gels, and Spectra™ multicolour broad range protein ladder was used as 153 the molecular mass marker. After electrolysis, gels were rinsed with distilled water and stained with 154 Coomassie brilliant blue R-250 for 3 h at 20 ℃. The activity of human cathepsins L, B, H, and K as influenced by the Lf hydrolysate was 159 measured using fluorometric inhibitor screening kits. The assay uses the ability of active human 160 cathepsin to cleave a synthetic substrate 7-amino-4-trifluoromethylcoumarin (AFC) and release a 161 fluorescent component that could be detected and measured using a fluorescence microplate reader.

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Cleavage of substrate is decreased in the presence of a cathepsin inhibitor, resulting in reduced or 163 no formation of AFC fluorescence. All cathepsin assays were performed in accordance with the 164 detailed protocols outlined in the Abcam booklet (www.abcam.com).

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For measuring the inhibitory efficacy on CTLS, a CTSL reagent aliquot was made by mixing 166 5 μL dithiothreitol (DTT) with CTSL reagent. According to the preliminary experiments, the 167 inhibitor compound was prepared by dissolving Lf hydrolysate (0-180 min hydrolysis time in 168 replicates) in CTSL assay buffer at final concentrations of 1.45 × 10 -8 , 6.25 × 10 -7 , 2.50 × 10 -6 , 7.50 169 × 10 -5 , and 2.50 × 10 -4 M. For measuring the inhibitory efficacy on other cathepsins (B, H, and K), a 170 slightly different protocol was applied, i.e., DTT was not used. The difference in protocol was reading for cathepsin L was 30 min, while that for cathepsin B, H, and K was 120 min.

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The activities of all cathepsins were monitored every 3 min in VersaMax microplate reader For spectra acquisition, the FTIR measurements were taken with a Bruker Invenio-S FTIR 185 spectrometer (Bruker Optik GmbH, Germany) and data acquisition was controlled by OPUS 8.5 186 software. The spectra were recorded in transmission mode from 4000 cm -1 to 400 cm -1 with a 187 resolution of 4 cm −1 , an aperture of 6.0 mm, and a total of 60 scans. The background and buffer 188 were collected before each sample scanning and subtracted at the same time, while 1% SDS and 189 Milli-Q water were used to clean the cell.

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For spectra analysis, the spectra were smoothed and normalised, and second-derivative 191 spectra were obtained by Savitsky-Golay derivative method for a nine data point window using 192 OPUS 8.5 software. Then the second derivative curves were cut from 1700 cm −1 to 1600 cm −1 193 (amide I) and analysed by fitting Gaussian curve using OriginPro 2021 software (OriginLab 194 Cooperation, Northampton, US). The quantification of secondary structures was performed by 195 calculating each peak area as a percentage of the sum of all peak areas.

Cathepsin inhibitory and secondary structure
The anti-cathepsin activity of bovine Lf hydrolysate samples was measured (Fig. 2)  short-term hydrolysis and generation of the half molecules (Fig. 1a)  proportion of these two structures was determined.

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Low α-helix content is a characteristic feature of many cysteine protease inhibitors such as 308 bromelain inhibitor VI (Hatano, Kojima, Tanokura, & Takahashi, 1995), and the cytotoxic T-309 lymphocyte antigen-2β (Delaria et al., 1994). Indeed, Lf was employed in the present study because 310 if its low α-helix content (Madadlou, 2020). Therefore, the percentile proportion of unordered-311 helical structures was measured and is reported in inhibitory activity was measured using screening assay kits (abcam.com), not by cell cultures and    Reference inhibitor 5.70 × 10 -9 a 8.14 × 10 -9 a 6.30 × 10 -9 a 9.50 × 10 -9 a a H0-H180 indicate bovine lactoferrin hydrolysed for different times in min. The reference inhibitor, Z-Phe-Phe-fluoromethyl ketone, is a synthetic inhibitor of cathepsins used in the cathepsin inhibitory assay. IC50 is the concentration of bovine lactoferrin hydrolysate required to achieve 50% cathepsin inhibition; the IC50 on pseudovirus is the concentration of bovine lactoferrin that decreases a single round of infection by 50%. The determination of secondary structure was based on the second-derivative amide I FTIR spectra. Different lowercase superscript letters indicate significant differences (p < 0.05) in a column (in either the cathepsin inhibition assay or pseudovirus inhibition assay); different uppercase superscript letters indicate significant differences (p < 0.05) in a row, in the cathepsin inhibition assay.
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