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Amani Alhalwani, John E. Repine, J. Alex Huffman; Nitration reduces lactoferrin antibacterial activity. Invest. Ophthalmol. Vis. Sci. 2017;58(8):489.
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© ARVO (1962-2015); The Authors (2016-present)
Lactoferrin (LF), a prominent component of tears and eye tissue, is a multifunctional protein that is a key contributor to ocular health and disease. Perturbations of LF develop in a variety of eye conditions such as infection, dry eye syndrome, and Sjögren’s Syndrome and, accordingly, may impact ocular function in many ways. LF perturbations undoubtedly occur as a consequence of ocular inflammation and exposure to environmental urban air pollutants, most notably, nitrating molecules, such as nitrogen oxide, that can nitrate tyrosine in proteins, like LF, on the surface of the eye. Because one important function of LF is antibacterial activity, we hypothesized that inflammation and environmental pollution inducing nitrating reactions could decrease the antibacterial activity of LF and contribute to ocular infection, dry eye syndrome, Sjorgren’s syndrome, and other ocular disorders. The specific aim of our study was to investigate the effect of nitration on the chemical structure and antibacterial function of lactoferrin.
Human lactoferrin (LF) was nitrated using two nitrating agents: tetranitromethane (TNM) mixed with methanol in a Tris-HCl buffer was used as a surrogate exogenous nitrating agent; peroxynitrite (ONOO-) in PBS buffer was used as a surrogate endogenous nitrating agent. LF chemical properties were then determined using absorbance spectroscopy, fluorescence, and SDS-PAGE. Subsequently, NLF concentrations were measured using a sandwich ELISA assay recently developed in our laboratory. Antimicrobial activity against Escherichia coli and Entercococcus faecalis of NLF was evaluated using the standard agar plate inhibition test.
We found that reaction with TNM and ONOO- changed the fundamental chemical properties of LF. Absorbance spectroscopy of LF revealed a peak at 280 nm, while, in contrast, following nitration, the absorbance peak of NLF increases to 350 nm. NLF also had a reduced fluorescence intensity compared to LF. Finally, NLF had reduced antibacterial activity compared to LF.
Nitration of lactoferrin produced in aqueous solution using TNM and ONOO-changed the chemical structure and reduced the antimicrobial activity of LF. Our results suggest that nitration of LF by exposure to inflammation and environmental nitrosative oxidants may contribute to ocular disorders such as infection, dry eye syndrome, and Sjögren’s Syndrome by reducing LF antibacterial and other protective activities.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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