Sensitization to cockroach allergens is a major risk factor for asthma, especially among inner city residents. The structure and function of the cockroach allergen Bla g 1 was determined by X-ray crystallography and NMR. The structure revealed a lipid binding protein, which is a common property of many allergens. However, the mechanism of action and the relative importance of the cargo lipids versus the allergen itself are poorly understood. In the past year the main goal was to generate forms of Bla g 1 engineered to either lack lipids, or bear a variety of different lipids of choice, including those found in its native form. Results have demonstrated that the protein can be stripped of its lipid cargo and reconstituted with a variety of lipid ligands. The plan is to test the potency of these various preparations in Dr. Cooks lab using a murine model of allergic sensitization through the airway. Further characterizing the lipid binding properties of Bla g 1, will be correlated with the allergic sensitization data to better understand how lipid adjuvants skew the immune response toward allergic sensitization. Gltuathione S-transferase (GST) allergens are found in many different species but these have received comparatively less attention than other more common allergenic folds. We investigated the cross-reactivity by determining the structures of Bla g 5, Der p 8, and Blo t 8, all GST-allergens. Further, we compared the cross reactivity of these allergens with a GST from another helminth, Ascaris sp., which we suspected might have similar cross reactivity to the other GST allergens. Using the structures we compared surface exposed residues and correlated the information with patient cross-reactivity. Despite published reports of cross-reactivity among GST allergens from patients in tropical countries there was very little cross-reactivity found in patients from N. Amercia, a more temperate biome, where the predominant GST allergen sensitizers are Bla g 5 and Der p 8. This result is substantiated by comparing the surface residues of the structures of all these allergens. There are very few regions where there is significant residue identity. This information is useful for clinicians. It informs them that N. American patients sensitized to Bla g 5 or Der p 8 are unlikely to be sensitive to the allergens from tropical species of mites or helminths. In addition, since the GST antibody response is species specific, identification of IgE to these allergens can be used to accurately diagnose the sensitizing species so that appropriate treatment can be prescribed. The protein Ara h 2 is the most potent peanut allergen recognized by >90% of peanut allergic patients. The natural allergen and the recombinant construct used to determine the structure showed different patterns of recognition by patient sera. Based on these comparisons a major site of interaction (an epitope) for about 50% of patients was identified. This success has encouraged us to further map the patient epitopes using a panel of antibodies with various specificities for Ara h 2 and the homologous Ara h 6 allergen. Over the past year the genes for the murine antibodies were cloned and we have created recombinant expression systems in E. coli and HEK cells that are being tested for production. It is our goal to further identify conformational epitopes on peanut allergens in order to better understand the patient response to peanut and to determine whether specific epitope recognition correlates with any aspect of peanut allergic disease, e.g. risk of anaphylaxis, emergency room visits, or response to oral therapy. Similar to the study above, we have generated an ScFv expression system for the anti-Der p 7 antibody WH9. This antibody blocks up to 60% of the patient response to the important dust mite allergen. In addition, antibodies to Bla g 5 were cloned and designed for expression systems. The various Bla g 5 antibodies span most of the protein surface and will be extremely useful in mapping patient epitopes. Improved understanding of the epitopes of the complex with Der p 7 or Bla g 5 will be facilitate better understanding the human response, with the hope of generating future therapeutics. Several previous studies, including those from our group, have identified the human protein RAGE as potentially important in the pathway of sensitization to allergens. We previously demonstrated that RAGE binds to peanut allergens Ara h 1 and Ara h 3 after the peanuts have been roasted. It has been proposed that the process of dry roasting contributes to sensitization and this provided direct mechanistic evidence that this was possible via the RAGE receptor. Further research proceeded by making improvements in the identification of the modifications to peanut proteins. This approach required a number of technical innovations, including computational strategies and isotopic labeling. Hopefully, this approach can shed light on the basis for the apparent ligand promiscuity of this receptor, which may lead to new approaches for inhibiting or enhancing this response in patients, depending on the desired outcome. It is widely accepted in allergen research that general features such as protein stability and abundance from source are common factors of allergens. However, rigorous statistical comparisons of allergens versus non-allergens on genomic and proteomic scales are lacking. In our study performed this year, the house dust mite Dermatophagoides pteronyssinus (DP) proteome was evaluated using RNA-seq methods as a proxy to assess the abundance of all proteins in this source. In addition, the thermodynamic stabilities of 700 non-allergens and 20 allergens were evaluated using a combined chemical denaturation and mass spectrometry approach. The results showed that when expression and stability are considered in combination, the allergens are a statistically different population from other DP proteins. The allergens are more stable and more highly expressed. Further research is needed to evaluate if this trend is true of other allergen sources. We are planning to interrogate cockroach and pollen extracts in a similar set of experiments. Understanding the biophysical properties of allergens is a major goal of this research. These molecular characterizations may be useful in evaluations of new GMOs to assess to potential for introduction of new allergenic proteins. Our studies on pollen extracts were motivated by previous work on ragweed and birch pollen that suggested the adenosine content was an important factor in allergic sensitization. This study compared the metabolite profile of 22 pollen species important for allergic disease, measured the adenosine content, and evaluated exposure to pollen-derived adenosine. A principal component analysis of the various metabolites identified by NMR showed that pollen extracts could be differentiated primarily by sugar content (glucose, fructose, and sucrose), and to a lesser extent glycerol, and myo-inositol. Adenosine was highest for grasses followed by trees and weeds. Pollen count data showed that tree pollen was typically 5-10 times the level of other pollens. At the daily peaks of tree, grass, and weed season the pollen-derived adenosine exposure per day is likely to only be 0.6, 0.04, and 0.07 g, respectively. We concluded that sugar content and other metabolites may be useful in classifying pollens. Unless other factors create exposures that are very different from the models we developed, pollen-derived adenosine is unlikely to be a major factor in allergic sensitization.
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