Oligomerization and Nitration of the Grass Pollen Allergen Phl p 5 by Ozone, Nitrogen Dioxide, and Peroxynitrite: Reaction Products, Kinetics, and Health Effects

The allergenic and inflammatory potential of proteins can be enhanced by chemical modification upon exposure to atmospheric or physiological oxidants. The molecular mechanisms and kinetics of such modifications, however, have not yet been fully resolved. We investigated the oligomerization and nitration of the grass pollen allergen Phl p 5 by ozone (O(3)), nitrogen dioxide (NO(2)), and peroxynitrite (ONOO(–)). Within several hours of exposure to atmospherically relevant concentration levels of O(3) and NO(2), up to 50% of Phl p 5 were converted into protein oligomers, likely by formation of dityrosine cross-links. Assuming that tyrosine residues are the preferential site of nitration, up to 10% of the 12 tyrosine residues per protein monomer were nitrated. For the reaction with peroxynitrite, the largest oligomer mass fractions (up to 50%) were found for equimolar concentrations of peroxynitrite over tyrosine residues. With excess peroxynitrite, the nitration degrees increased up to 40% whereas the oligomer mass fractions decreased to 20%. Our results suggest that protein oligomerization and nitration are competing processes, which is consistent with a two-step mechanism involving a reactive oxygen intermediate (ROI), as observed for other proteins. The modified proteins can promote pro-inflammatory cellular signaling that may contribute to chronic inflammation and allergies in response to air pollution

Simultaneous determination of nitrated and oligomerized proteins by size exclusion high-performance liquid chromatography coupled to photodiode array detection

Chemical modifications such as nitration and cross-linking may enhance the allergenic potential of proteins. The kinetics and mechanisms of the underlying chemical processes, however, are not yet well understood. Here, we present a size-exclusion chromatography/spectrophotometry method (SEC-HPLC-DAD) that allows a simultaneous detection of mono-, di-, tri-, and higher protein oligomers, as well as their individual nitration degrees (NDs). The ND results of proteins from this new method agree well with the results from an alternative well-established method, for the analysis of tetranitromethane (TNM)- and nitrogen dioxide and ozone (NO2/O3)-nitrated protein samples. Importantly, the NDs for individual oligomer fractions can be obtained from the new method, and also, we provide a proof of principle for the calculation of the concentrations for individual protein oligomer fractions by their determined NDs, which will facilitate the investigation of the kinetics and mechanism for protein tyrosine nitration and cross-linking

Determination of nitration degrees for the birch pollen allergen Bet v 1

Nitration of tyrosine residues in the major birch pollen allergen Bet v 1 may alter the allergenic potential of the protein. The kinetics and mechanism of the nitration reaction, however, have not yet been well characterized. To facilitate further investigations, an efficient method to quantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) that can be photometrically calibrated using the amino acids tyrosine (Tyr) and nitrotyrosine (NTyr) without the need for nitrated protein standards. The new method is efficient and in agreement with alternative methods based on hydrolysis and amino acid analysis of tetranitromethane (TNM)-nitrated Bet v 1 standards as well as samples from nitration experiments with peroxynitrite. The results confirm the applicability of the new method for the investigation of the reaction kinetics and mechanism of protein nitration

Chemical modification of aeroallergens Bet v 1 and Phl p 5 by air pollutants and physiological peroxynitrite affects serum IgE binding and TLR4 signaling

Asthma and allergy are major health problems in most modern so-cieties, and numerous studies indi-cate that allergic diseases have been increasing during the past decades. The growing prevalence of allergies in industrialized countries may be linked, beside other factors, to air pollution. Allergenic proteins are efficiently nitrated and cross-linked by atmospheric ozone and nitrogen dioxide (O3/NO2) or physiologically relevant peroxynitrite (ONOO-), and the modifications occur primarily at tyrosine residues. In several studies, modified food and airborne allergens showed an altered allergenic potenti-al, but the underlying chemical and immunological mechanisms remain unclear. Here, the major birch pollen allergen Bet v 1 and the major grass pollen allergen Phl p 5 were exposed to atmospherically relevant concent-rations of ozone and nitrogen dioxide (O3/NO2) and different levels of per-oxynitrite (ONOO-). To determine the degree of tyrosine nitration and tyrosine cross-linked dimeric and oligomeric allergens, reversed-phase (C18) and size-exclusion chromato-graphy (SEC) coupled to a diode ar-ray detector (DAD), and SDS-PAGE were used. IgE binding ELISA using sera of birch and grass pollen allergic patients assessed altered immunity of the modified allergens. To study parts of the innate immune respon-se, TLR4 activation were measured using respective reporter cell lines. Modified Bet v 1 (ONOO-) leads to higher IgE binding in almost all pati-ents, whereas modified Phl p 5 does not show higher IgE binding. In con-trast, ONOO- modification of Phl p 5 leads to higher TLR4, while modified Bet v 1 induced no activation. Thus, modification of Bet v 1 seems to al-ter IgE epitopes, which is in line with earlier studies, whereas the modifica-tion of Phl p 5 affects innate immu-ne reactions, rather than IgE epitope binding. Therefore, the two allergens alter immune reactions by different modes of actions, either by the innate or adaptive part of immunity

Determination of the protein content of complex samples by aromatic amino acid analysis, liquid chromatography-UV absorbance, and colorimetry

Fast and accurate determination of the protein content of a sample is an important and non-trivial task of many biochemical, biomedical, food chemical, pharmaceutical, and environmental research activities. Different methods of total protein determination are used for a wide range of proteins with highly variable properties in complex matrices. These methods usually work reasonably well for proteins under controlled conditions, but the results for non-standard and complex samples are often questionable. Here, we compare new and well-established methods, including traditional amino acid analysis (AAA), aromatic amino acid analysis (AAAA) based on the amino acids phenylalanine and tyrosine, reversed-phase liquid chromatography of intact proteins with UV absorbance measurements at 220 and 280 nm (LC-220, LC-280), and colorimetric assays like Coomassie Blue G-250 dye-binding assay (Bradford) and bicinchoninic acid (BCA) assay. We investigated different samples, including proteins with challenging properties, chemical modifications, mixtures, and complex matrices like air particulate matter and pollen extracts. All methods yielded accurate and precise results for the protein and matrix used for calibration. AAA, AAAA with fluorescence detection, and the LC-220 method yielded robust results even under more challenging conditions (variable analytes and matrices). These methods turned out to be well-suited for reliable determination of the protein content in a wide range of samples, such as air particulate matter and pollen

Metaproteomic analysis of atmospheric aerosol samples

Metaproteomic analysis of air particulate matter provides information about the abundance and properties of bioaerosols in the atmosphere and their influence on climate and public health. We developed and applied efficient methods for the extraction and analysis of proteins from glass fiber filter samples of total, coarse, and fine particulate matter. Size exclusion chromatography was applied to remove matrix components, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied for protein fractionation according to molecular size, followed by in-gel digestion and LC-MS/MS analysis of peptides using a hybrid Quadrupole-Orbitrap MS. Maxquant software and the Swiss-Prot database were used for protein identification. In samples collected at a suburban location in central Europe, we found proteins that originated mainly from plants, fungi, and bacteria, which constitute a major fraction of primary biological aerosol particles (PBAP) in the atmosphere. Allergenic proteins were found in coarse and fine particle samples, and indications for atmospheric degradation of proteins were observed

Water uptake of subpollen aerosol particles: hygroscopic growth, CCN activation, and liquid-liquid phase separation

Pollen grains emitted from vegetation can release subpollen particles (SPP) that contribute to the fine fraction of atmospheric aerosols and may act as cloud condensation nuclei (CCN), ice nuclei (IN), or aeroallergens. Here, we investigate and characterize the hygroscopic growth and CCN activation of birch, pine, and rapeseed SPP. A high humidity tandem differential mobility analyzer (HHTDMA) was used to measure particle restructuring and water uptake over a wide range of relative humidity (RH) from 2 % to 99.5 %, and a continuous flow CCN counter was used for size-resolved measurements of CCN activation at supersaturations (S) in the range of 0.2 % to 1.2 %. For both, subsaturated and supersaturated conditions, effective hygroscopicity parameters κ, were obtained by Köhler model calculations. Gravimetric and chemical analyses, electron microscopy, and dynamic light scattering measurements were performed to characterize further properties of SPP from aqueous pollen extracts such as chemical composition (starch, proteins, DNA, and inorganic ions) and the hydrodynamic size distribution of water-insoluble material. All investigated SPP samples exhibited sharp increases of water uptake and κ above ~95 % RH, suggesting a liquid-liquid phase separation (LLPS). The HHTDMA measurements at RH > 95 % enable closure between the CCN activation at water vapor supersaturation and hygroscopic growth at subsaturated conditions, which is often not achieved when HTDMA measurements are performed at lower RH where the water uptake and effective hygroscopicity may be limited by the effects of LLPS. Such effects may be important not only for closure between hygroscopic growth and CCN activation but also for the chemical reactivity, allergenic potential, and related health effects of SPPs