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.

中文翻译：

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过氧亚硝酸盐对乳铁蛋白的修饰降低了其抗菌活性并改变了蛋白质结构。

乳铁蛋白 (LF) 是一种多功能蛋白质，作为许多人类和其他哺乳动物体液和组织中浓度最高的蛋白质之一，在生理学方面发挥着重要作用。特别是，LF 为人乳、唾液和泪液提供抗菌特性。LF 还通过其铁结合能力在炎症部位防止应激诱导的脂质过氧化。先前的研究表明，LF 可以通过生物相关介质（如过氧亚硝酸盐 (ONOO-)）有效硝化，在炎症和亚硝化应激期间，过氧亚硝酸盐 (ONOO-) 也以高细胞内浓度存在。在这里，我们检查了 ONOO 处理后 LF 的抗菌性能和结构的变化。该反应诱导酪氨酸和色氨酸残基的硝化，这些残基通常用作多种疾病的生物标志物分子。用 ONOO- 以 10/1 M 的 ONOO- 与酪氨酸的比例处理抑制了天然 LF 表现出的所有抗菌活性。LF 的二级结构变化使用圆二色光谱法进行评估。添加和不添加 Fe3+ 的硝化产物显示出 α-螺旋特性的显着降低，表明部分蛋白质展开。用 ONOO- 处理后 LF 的铁结合能力也降低，表明 LF 防止细胞损伤的能力降低。LC-MS/MS 光谱法用于鉴定被 ONOO-硝化的 LF 肽片段，包括位于铁结合区的酪氨酸残基 Y92。这些结果表明，ONOO-对LF的翻译后修饰可能是加剧感染的重要途径，例如，