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Structural and Functional Characterization of Allergens

London, Robert
DHHS/NIH - National Institute of Environmental Health Sciences
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This project has succeeded in determining the structure of the Ara h 2 allergen from peanuts and mapping a major patient reactive site to the structure. The project has continued work on the Der p 7 allergen to further elucidate the natural ligand and better understand which pathways the allergen and natural ligand are likely to interact with in humans. A project was initiated to determine the structure of the cockroach allergen Bla g 1. The significance of each project is discussed below.Der p 7- More than 50% of house dust mite allergic patients react specifically to the protein Der p 7. It has been proposed that the natural function of many house dust mite allergens may be related to the generation of the allergic response. However, the function of Der p 7 could not be determined from sequence information alone.

The structure of Der p 7 determined by X-ray crystallography revealed an elongated structure with two 4-stranded beta sheets that wrap around a long C-terminal helix. This fold is highly similar to human proteins involved in the human innate immune response to bacteria. We have demonstrated that Der p 7 can bind to a compound derived from gram positive bacteria, which suggests that Der p 7 could skew the normal human response by interfering with the recognition of foreign lipid-like compounds. Therefore it would be very interesting to further characterize the types of compounds to which Der p 7 binds.

Our current efforts are focused on NMR based screening of compounds that are suggested either by the literature, or on the basis of computer simulations. A fragment library screen, like those performed by drug companies, was performed and has generated some promising leads. Knowing the class of ligands should provide more insight into what human pathways are modulated by these allergens and why the allergic response is so potent for dust mite allergens and Der p 7.Ara h 2- The protein Ara h 2 is the most potent peanut allergen recognized by >90% of peanut allergic patients. However, the structure of this important allergen had not previously been determined. We found that the structure is a 5 helix bundle held together by 4 disulfide bonds and related to the prolamin protein superfamily. 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. It is anticipated that the structure and this major epitope will provide information for the rational design of Ara h 2 hypoallergens for improved and safer immunotherapies.Bla g 1- We have initiated a new project to analyze the structure of the cockroach allergen Bla g 1. Sensitivity to cockroach allergens is strongly associated with asthma especially among patients with low socio-economic status. Bla g 1 is one of two allergens commonly used as markers of cockroach allergen exposures in environmental studies.

The function of Bla g 1 is unknown based on sequential information, and similar to our studies of Der p 7, we hypothesize that analysis of the structure will help understand the function of the protein and help identify human pathways that cockroach allergens interfere with. Identifying these human pathways will hopefully lead to new or improved treatments of allergic disease.

Currently we have scaled up production of the protein in order to perform initial crystallization experiments and NMR labeling. In addition, we obtained an E. coli expressible antibody fragment (ScFv) against Bla g 1. To date we have engineered the ScFv for large scale production and demonstrated that is does bind to our Bla g 1 constructs. The ScFv should be useful as a co-crystallization agent, and may be useful in identifying patient epitopes on Bla g 1.

Funding Source
Nat'l. Inst. of Environmental Health Sciences
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Escherichia coli
Nuts, Seeds