Detailed accomplishments of each project are summarized below. 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 one 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. Purification and scale up procedures are being developed to this end. The plan is to test the potency of these various preparations in Dr. Cooks lab by 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. A recent paper sparked renewed interest in these allergens due to the demonstrated cross-reactivity of the GST allergen Bla g 5 with a helminth GST from Wucheria bancrofti. A connection between the immune response to helminth and cockroach allergens has long been suspected because humans respond to both with same antibody subtype, IgE. We investigated the cross-reactivity further 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. Currently we have selected and produced the Fab fragment of several antibodies and are currently performing crystallization trials with complexes of Ara h 2. Thus far, we have failed to generate quality crystals. We plan to attempt new strategies for crystallization including C-terminal affinity/crystallization tags for Ara h 2. Another problem is the expense of production of the antibodies in hybridomas for the quantities needed for crystallization. Over the past year the genes for the 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 specifically 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 is proceeding in two directions. First, the modifications to the peanut allergens are being more carefully categorized by mass spectroscopy. This approach required a number of technical innovations, including computational strategies and isotopic labeling. We are beginning to categorize the modifications using a more significant sample size. Remarkably, we have found that RAGE is capable of recognizing the diverse array of chemical modifications from cooking. We are attempting to synthesize specific modifications on peanut allergen-derived peptides in order to characterize how these modifications interact with RAGE. 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.
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