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.