Impact of nitration on air-borne allergens

Previous research has shown that nitration of allergens can occur following exposure to ozone, nitric oxide and water in polluted environments. Moreover, allergens might be nitrated by reactive oxygen species in inflamed lung tissues. The nitration of proteins mainly affects the tyrosine residues, resulting in the formation of nitrotyrosine. The specificity of this modification can be related to a multitude of factors, such as the position of the tyrosine residue, the secondary structure of the protein and the nitration agent. However, the exact mechanism behind nitration events under natural conditions is still unknown. For research purposes, tetranitromethan is used to nitrate proteins in vitro and determine the biological effects of these modified proteins in comparison to the naive proteins. Over the past years, we have studied the effects of Bet v 1a (the major birch pollen allergen) nitration on the immunogenicity of this protein. Thus far, we could show that the serum from patients with an existing birch pollen allergy have IgE specific antibodies that are specific for the nitrated Bet v 1a and that do, at least in part, not cross react with normal Bet v 1a. It is therefore likely that the nitration of Bet v 1a results in the expression of novel epitopes that can result in nitrated Bet v 1a specific antibody production. In a subsequent study, it was shown that the presentation of nitrated Bet v 1a derived peptides by dendritic cells is increased and that the diversity in nitro- Bet v 1a epitopes is enhanced (when compared to normal Bet v 1a) and that this increased presentation results in an increased T cell activation. These results implicate that nitration leads to protein alterations, resulting in an increased uptake or better intracellular digestion. So far, characterisation of the nitrated Bet v 1a has shown that the modification resulted in protein multimerisation. Preliminary data with the grass pollen allergen Phl p 5 showed that the same effects did not occur for this allergen. We therefore postulate that nitration of proteins is a selective procedure that might not affect all allergens to the same extent. Our main goal in the proposed study will be to determine the effects of allergen nitration on the protein itself and on immune reactions induced by these modified proteins; this study can be divided in three parts. First of all, we want to study whether allergy patients have in general enhanced levels of IgE specific antibodies to the disease specific nitrated allergens compared to unmodified allergens. Secondly, the effects of nitration on the conformation and aggregation of different allergens will be investigated. In the final part, the effect of the nitration grade on the immunological and chemical factors described above will be addressed and these levels will be compared with those found for natural allergens and allergens that have been nitrated under physiological circumstances. The latter study will focus on birch pollen allergens. The results of the proposed study will not only enhance our knowledge with respect to the effects of protein nitration on allergic responses, but will also provide information on protein nitration in general. Moreover, the study will initiate the unravelling of the most likely mechanisms for protein nitration and the effects of such a nitration on protein structure, folding and interactions. The inclusion of several different allergens will increase the knowledge regarding the specificity of nitration and impact of these specific chemical modifications.

The number of patients suffering from an allergy in industrialised countries has increased over the past decades. The reasons behind this increase are still unknown, but several plausible explanations are currently studied. Previously, scientists discovered that plant pollen in street dust can be chemically modified in the presence of high levels of ozone and nitric oxide in polluted air. Under these conditions, nitration was the most frequent chemical modification which resulted in a very specific modification of the allergens. These findings triggered us to study the effects of allergen nitration on allergic responses. Previous studies in our laboratory showed, that nitration of allergens could enhance the immune responses against these modified proteins. However, the exact mechanisms behind the alterations are unknown. The current study therefore aimed at a closer characterisation of nitrated allergens on different levels. For these studies, the birch pollen allergen Bet v 1.0101 was taken as a model protein. Immunological responses were analysed using different human cell-based systems. Such systems allow us to work with human material and avoid the use of animals for laboratory experiments. The immunological data showed that the inflammation-promoting branch of the immune system was inhibited when different immune cells were brought in contact with nitrated allergens. Since the allergy and the inflammation branch of our immune system should be in balance, an inhibition of inflammation promotes allergic responses. Several different methods for protein nitration were tested in close collaboration with the Max-Planck Institute (Mainz, Germany). One method applied a nitration reaction that is routinely used in laboratories (tetranitromethane). A second method was related to nitration in the environment (as described above) and the third method mimicked the situation in inflamed lung tissue. The classical nitration method turned out to be a good representative for the more biological nitration methods, but in the air and in inflamed tissue, oxidation plays a very important role besides nitration and this could also affect the proteins. The second part of the project was dedicated to the analysis of the structural changes and the differentiation between numerous nitrated species that occur after the nitration of allergens. Many analytical methods require the proteins to be cleaved into small pieces before analysis. This destruction of the structure leads to a loss of information. In our study, capillary electrophoresis with UV and mass spectrometric detection was used to allow for the analysis of intact proteins. New analytical methods were successfully established and applied including an in-lab designed interface for mass spectrometry. Several different biochemical techniques could be interlinked in order to allow for a very precise determination of the protein structure and to monitor the changes that were induced by nitration. One of the findings was that nitrated allergens often formed stable agglomerates, so called multimers, and multimeric proteins are known to stimulate immune responses.