Modification of lactoferrin by peroxynitrite reduces its antibacterial activity and changes protein structure

Abstract

Lactoferrin (LF) is a multifunctional protein that plays important physiological roles as one of the most concentrated proteins in many human and other mammalian fluids and tissues. In particular, LF provides antibacterial properties to human milk, saliva, and tear fluid. LF also protects against stress‐induced lipid peroxidation at inflammation sites through its iron‐binding ability. Previous studies have shown that LF can be efficiently nitrated via biologically relevant mediators such as peroxynitrite (ONOO−), which are also present at high intracellular concentrations during inflammation and nitrosative stress. Here, we examine changes in antibacterial properties and structure of LF following ONOO− treatment. The reaction induces nitration of tyrosine and tryptophan residues, which are commonly used as biomarker molecules for several diseases. Treatment with ONOO− at a 10/1 M ratio of ONOO− to tyrosine inhibited all antibacterial activity exhibited by native LF. Secondary structural changes in LF were assessed using circular dichroism spectroscopy. Nitration products with and without the addition of Fe3+ show significant reduction in alpha‐helical properties, suggesting partial protein unfolding. Iron‐binding capacity of LF was also reduced after treatment with ONOO−, suggesting a decreased ability of LF to protect against cellular damage. LC‐MS/MS spectrometry was used to identify LF peptide fragments nitrated by ONOO−, including tyrosine residue Y92 located in the iron‐binding region. These results suggest that posttranslational modification of LF by ONOO− could be an important pathway to exacerbate infection, for example, in inflamed tissues and to reduce the ability of LF to act as an immune responder and decrease oxidative damage.

Tear Film Proteome of Healthy Domestic Cats

“…is finding corroborates the literature, which cites the presence of the protein in question in the TF of humans [44], koalas [14], and dogs [14,52]. It is a multifunctional binding protein [29], produced by the acinar cells of the main and accessory lacrimal glands, with antimicrobial and anti-inflammatory action. erefore, it plays an important role in innate immunity and helps maintain ocular surface homeostasis [3,29].…”

“…It is a multifunctional binding protein [29], produced by the acinar cells of the main and accessory lacrimal glands, with antimicrobial and anti-inflammatory action. erefore, it plays an important role in innate immunity and helps maintain ocular surface homeostasis [3,29]. Its antimicrobial action occurs because lactotransferrin inhibits the classical complement activation pathway and binds to free iron in tears, reducing the availability of iron required for bacterial growth [3,29,43].…”

“…erefore, it plays an important role in innate immunity and helps maintain ocular surface homeostasis [3,29]. Its antimicrobial action occurs because lactotransferrin inhibits the classical complement activation pathway and binds to free iron in tears, reducing the availability of iron required for bacterial growth [3,29,43].…”

Human Milk Antioxidative Modifications in Mastitis: Further Beneficial Effects of Cranberry Supplementation

“…Furthermore, as mentioned above, recent studies have reported that mastitis increases LF concentrations [ 27 , 50 ]. LF sequesters Fe 3+ , preventing the formation of hydroxyl radicals via the Fenton reaction, and in turn prevents lipid peroxidation [ 51 , 60 ]. Therefore, the increase in LF content in milk could also contribute to the decrease in MDA concentration observed in the mastitis group ( Figure 4 a,b).…”

Reactive Nitrogen Species in Brain After in Vivo Exposure to Arsenic

“…At this time, it is proposed that the main cause, at least for protein modification, is related to the reactive nitrogen species (RNS) production. These species are generated when NO reacts with O 2 to form peroxynitrite (ONOO -), a strong oxidant, that easily reacts with nucleophilic molecules (Alhalwani et al, 2020). If the production of NO is increased, the steady state concentration of ONOOis also enhanced, and this situation could harm proteins via tyrosine (Tyr) nitration (Radi, 2018).…”