A Peptide Bond from the Inter-lobe Segment in the Bilobal Lactoferrin Acts as a Preferred Site for Cleavage for Serine Proteases to Generate the Perfect C-lobe: Structure of the Pepsin Hydrolyzed Lactoferrin C-lobe at 2.28 Å Resolution

Lactoferrin: A glycoprotein that plays an active role in human health

“…In addition, the N-lobe and C-lobe are connected by a small peptide bond (amino acids 334-344), forming the overall structure as “two Ginkgo biloba type,” with each lobe having an iron-binding site in the cleft between the N1 and N2, C1 and C2 sub-structural domains. What’s more, iron ions are covalently linked to four amino acid residues (two tyrosine, one aspartate and one histidine) of Lf ( 22 ). In the presence of carbonate, each lobe is able to reversibly bind Fe 3+ with a maximum binding amount of 1.4 mg Fe/g protein.…”

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

“…The production of Lf half molecules due to hydrolysis confirms that proteinase K cuts Lf at the helix that covalently connects two lobes of Lf ( Singh, Sharma, Karthikeyan, Betzel, & Bhatia, 1998 ). The peptide bond Arg341–Tyr342, which is a part of the inter-lobe 10-residue long helical peptide Thr334-Thr343 is cleaved by proteinase K ( Singh et al, 2021 ). Under non-reducing SDS-PAGE, several bands at ≤18 kDa were observed in all the hydrolysate samples (≥30 min).…”

“…1 a). The small fragments (≤18 kDa) were most probably the hydrolysis products of the N-lobe and the C-lobe supposedly remained fully functional ( Singh et al, 2021 ). The band at ∼12 kDa became faint by hydrolysis ≥60 min.…”

“…Based on the results of a molecular docking early assessment which revealed that only the C-terminal lobe of Lf interacts with CTSL ( Madadlou, 2020 ), it was hypothesised that cutting bovine Lf molecules to the half-molecules may be advantageous for CTSL inhibition. Either of the enzymes pepsin, trypsin or proteinase K can be used to generate peptide fragments of half the molecular mass of Lf ( Sharma, Singh, & Bhatia, 1999 ; Singh et al, 2021 ). The objective of the current research was to hydrolyse bovine Lf by proteinase K and assess the inhibitory effect of the bovine Lf hydrolysate on the human cathepsins L, B, H, and K. Next, SARS-CoV-2 pseudovirus model tests were performed to determine whether the CTSL-inhibitory bovine Lf hydrolysate could prevent the infection of human cells under in vitro conditions.…”