Lactoferrin (LF) is an important ocular protein with polyfunctional properties,such as anti-inflammatory. Inflammation can enhance production of nitrated proteins, leading to changes in protein structure and functions. Lactoferrin can be nitrated by common processes within the body, yet few studies have investigated the properties of chemically modified LF. Our goal of study was to investigate the effect of nitration on the antibacterial function of LF by testing the function of nitrated lactoferrin (NLF) by an antibiotic susceptibility test to measure the loss of antibacterial activity upon nitration,circular dichroism (CD) to measure changes in secondary protein structure,and iron binding assay to measure the effect on this critical LF function. Human lactoferrin was nitrated using peroxynitrite (ONOO-) and produced nitrated lactoferrin.The structure of NLF vs. LF was investigated using CD to determine the secondary structure. The iron binding capacity of NLF was tested using a ferric nitrilotriacetic acid solution, with detection by absorbance. The antimicrobial activity of LF vs. NLF was evaluated against Escherichia coli (E. coli) using a broth susceptibility test. The nitration reaction by ONOO- changed the chemical structure of LF, with increasing effect at higher nitration ratios. The antibacterial activity of LF was reduced after nitration. The measured absorbance after assay testing was 0.38 ± 0.04 for untreated E. coli, 0.31 ± 0.02 for LF, and 0.37 ± 0.04 for NLF, suggesting that upon nitration LF loses nearly 100% of its antibacterial activity. The t-test showed the difference in the activity of NLF compared to LF to be significant p-value &lt; 0.001. The iron binding assay showed a minor reduction in binding after nitration. The CD spectra of NLF show a major change in secondary structure compared to LF. Further test will be needed to understand the structural changed account for decreased antibacterial activity of LF by nitration. Our results suggest that NLF provides reduced antimicrobial activity compared to LF, due to changes in the molecular structure rather than due to changes at the iron-binding site. These results are the first to show the effect that nitration can play in reducing antimicrobial activity in human fluids. The combination of CD and iron-binding results also provide the first evidence for a mechanism of reduction of antimicrobial activity due to changes in protein structure.

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

2019 Fall National Meeting

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