Structure of allergens

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Allergies


IgE-mediated allergies represent a major health problem in the industrialized world. The immediate symptoms of the disease (e.g. allergic rhinoconjunctivitis, dermatitis, bronchial asthma, anaphylactic shock) are caused by the cross-linking of effector cell-bound IgE antibodies by allergens which leads to the release of biological mediators such as histamine or leukotriens). In order to induce strong effector cell activation and thus inflammatory responses, an allergen must be able to cross-link effector cell-bound IgE antibodies efficiently. This process requires the presence of at least two IgE epitopes on the allergen surface. IgE antibodies of allergic patients may either recognize "continuous epitopes" consisting of a row of consecutive amino acids or "discontinuous epitopes" which are composed of amino acids from different portions of the allergen brought into proximity by the fold of the molecule. The 3D structures of allergens are essential for the precise analysis of IgE-epitopes.
The aim of our project is the structure elucidation of major environmental allergens as a basis for rational design of hypoallergenic derivatives for vaccine development.This project is part of the new SFB F018, funded by the Austrian national science foundation FWF:

A schematic overview of the allergy mechanism - click to see more details


Molecular and Immunological Strategies for Prevention, Diagnosis and Treatment of Type I Allergies




Calcium-Binding Allergens


A photo of the timothy grass (Phleum pratense), a sorce of grass pollen allergens - click to see more details The structure of Phl p 7, a calcium binding pollen allergen from timothy grass - click to see more details




The first calcium-binding allergen, parvalbumin, has been characterized more than 30 years ago. This major fish allergen is a small acidic calcium buffer protein in fast-twitch muscle that can be easily purified from the natural sources by boiling of crude muscle extracts. Since then, a great number of allergen sequences have become available due to the introduction of molecular biology techniques for allergen characterization. Sequence analysis of allergen-encoding cDNAs revealed the presence of the typical calcium-binding EF-hand motifs within many allergens. Up to date calcium-binding allergens have been isolated from plants, parasites, fish and man. Most of these proteins are expressed preferentially in certain tissues.
Most calcium-binding allergens described so far represent low-molecular weight, water-soluble proteins. It was shown that several representatives exhibit great stability when exposed to heat, denaturing agents or enzymes, a fact that may be related to their allergenicity. The calcium-bound forms of parvalbumin and two EF-hand allergens such as Phl p 7 and Aln g 4, showed a higher stability than the apo-forms, indicating that calcium greatly contributes to overall proteins stability.
Calcium-binding allergens may also be grouped according to immunological cross-reactivity. The two major cross-reactive allergens families include parvalbumins from various fish species (cod, salmon, carp) and the two EF-hand pollen allergens from trees, grasses and weeds. There is no experimental evidence for IgE cross-reactivity between the EF-hand allergens from plants, fish, schistosomes and man. This finding suggests that the major IgE epitopes do not reside in the calcium-binding loops which are conserved among all calcium-binding proteins but rather comprise the less-conserved portions of the proteins.





Circular Dichroism Spectroscopy


CD spectroscopy proves to be a useful technique for examining the structure of biological macromolecules (i.e. proteins, peptides and nucleic acids) in solution under different conditions and for examining both their dynamics and folding pathways. Secondary and tertiary structures can be monitored by the peptide transitions in the far UV (~190-220 nm) and by the aromatic side chain transitions in the near UV (~270-290 nm), respectively. Examination of the effects of different pHs, detergents, solvents or other additives can be a guide to the integrity of the protein and "physiological relevance" of, for example, the crystallization conditions. Finally, thermal denaturation studies monitored by CD result in knowledge on the protein's (un)folding curves and can be used to establish means of enhancing the stability of the protein by addition of various types of additives.


CD spektra of Phl p 7 under calcium-depletion upon thermal denaturation



Thermal denaturation of PHL p 7 in the presence of EGTA monitored via CD-spectroscopy


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