Abstract
Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO–), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O3/NO2). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO–, and Y83 and Y158 for O3/NO2. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O3/NO2 was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein.
Highlights
Post-translational modifications such as nitration and oxidation of proteins can occur during inflammation, oxidative stress, and chemical aging under physiological or environmental conditions
Our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may affect the biological function and adverse health effects of the nitrated protein
Numerous studies have suggested that asthma and allergic diseases are enhanced by traffic-related air pollution with high concentrations of nitrogen oxides (NOx) and ozone (O3).[6,7]
Summary
Post-translational modifications such as nitration and oxidation of proteins can occur during inflammation, oxidative stress, and chemical aging under physiological or environmental conditions. Protein nitration has been reported in association with at least 50 different diseases,[1,2] and it has been shown to alter the immunogenic potential of food allergens[3] and aeroallergens like Bet v 1.4,5. Numerous studies have suggested that asthma and allergic diseases are enhanced by traffic-related air pollution with high concentrations of nitrogen oxides (NOx) and ozone (O3).[6,7] Nitration reactions and related changes in the immunogenicity of allergenic proteins might explain the promotion of allergies by traffic-related air pollution.[8] Recently, it has been shown that birch pollen from urban areas had a higher allergenic potential than pollen from rural areas, the allergen content remained unchanged,[9] and ragweed pollen collected along roads with heavy traffic showed a higher allergenic potential compared with pollen collected in remote areas.[10]. The nitration mechanism may either involve ionic species, formed from a partial dissociation of TNM into nitronium ions and nitroformate ions, or radical species.[11−13]
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