Inhibitory Effect of Bovine Lactoferrin on Catechol-O-Methyltransferase.

Masayuki Ikeda,Ichizo Shinoda,Hiroshi Iijima,Yasuhiro Takeda,Hiroshi Iwamoto

doi:10.3390/molecules22081373

Abstract

Lactoferrin (LF) is a well-known multifunctional protein. In this study, we report the inhibitory potency of bovine LF (bLF) on catechol-O-methyltransferase (COMT), which catalyzes methylation of catechol substrates. We found that bLF binds to and inhibits COMT using its N-terminal region. An N-terminal peptide fragment obtained from bLF by trypsin digestion showed a higher inhibitory activity than intact bLF. A synthetic fragment of the bLF N-terminal residues 6–50, with two pairs of disulfide bonds, also showed higher inhibitory activity than intact bLF. Enzyme kinetic studies proved that bLF did not compete with S-adenosylmethionine (the methyl donor substrate) as well as methyl acceptor substrates such as dihydroxybenzoic acid, (−)-epicatechin, norepinephrine, or l-3,4-dihydroxyphenylalanine. The inhibitory potency of bLF decreased against a COMT preparation pretreated with dithiothreitol, suggesting that the oxidation status of COMT is relevant to interaction with bLF. We further confirmed that COMT activity in the cell extracts form Caco-2 and HepG2 cells was inhibited by bLF and by the synthesized fragment. Enzyme kinetic study indicated that bLF functions as a non-competitive inhibitor by binding to an allosteric surface of COMT.

Highlights

Lactoferrin (LF) is a multifunctional protein distributed in body fluids such as milk and mucus, in addition to secondary granules of white blood cells [1,2,3]
P36 and P5 were more active than intact bovine lactoferrin (bLF), whereas lactoferricin B (LFcinB) was less active than intact bLF
The interaction site of bLF is located in the P5 region and existence of the two sulfide bonds is important. These disulfide bonds should constrain the flexibility of P5 and stabilize the active conformation that interacts with COMT

Summary

Introduction

Lactoferrin (LF) is a multifunctional protein distributed in body fluids such as milk and mucus, in addition to secondary granules of white blood cells [1,2,3]. LF is a ferric-binding protein and shows enhanced antibacterial activity by chelating ferric ions. Shows antibacterial activity [8]. LF binds to proteins such as intelectin [9], low-density lipoprotein receptor-related protein [10], nucleolin [11], Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [12], calmodulin [13], and lipopolysaccharide [14]. These properties may facilitate LF’s multifunctionality, and because it is a milk protein and is habitually ingested with other foods, its function is complex

Results

Discussion

Conclusion