Brigitte Hayek

Calcium–Binding Allergens: From Plants to Man

Calcium–binding proteins contain a variable number of motifs, termed EF–hands, which consist of two perpendicularly placed α–helices and an interhelical loop forming a single calcium–binding site. Due to their ability to bind and transport calcium as well as to interact with a variety of ligands in a calcium–dependent manner, they fulfill important biological functions in eukaryotic cells. After parvalbumin, a three EF–hand fish allergen, calcium–binding allergens were discovered in pollens of trees, grasses and weeds and, recently, as autoallergens in man. Although only a small percentage of atopic individuals displays IgE reactivity to calcium–binding allergens, these allergens may be important because of their ability to cross–sensitize allergic individuals. Conformation and stability as well as IgE recognition of calcium–binding allergens greatly depend on the presence of protein–bound calcium ions. It is thus likely that hypoallergenic derivatives of calcium–binding allergens can be engineered by recombinant DNA technology for immunotherapy of sensitized patients.

Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind‐pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF‐hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen‐specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7‐homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium‐bound and apo‐rPhl p 7 indicated that differences in IgE recognition may be due to calcium‐induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation‐dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.—Niederberger, V., Hayek, B., Vrtala, S., Laffer, S., Twardosz, A., Vangelista, L., Sperr, W. R., Valent, P., Rumpold, H., Kraft, D., Ehrenberger, K., Valenta, R., Spitzauer, S. Calcium‐dependent immunoglobulin E recognition of the apo‐ and calcium‐bound form of a cross‐reactive two EF‐hand timothy grass pollen allergen, Phl p 7. FASEB J. 13, 843–856 (1999)

Immunogold Electron Microscopic Localization of the Cross–Reactive Two–EF–Hand Calcium–Binding Birch Pollen Allergen Bet v 4 in Dry and Rehydrated Birch Pollen

Background: Recently, a novel family of low–molecular–weight (8–9 kD), two–EF–hand calcium–binding proteins has been described as allergens in plant pollens. Approximately 10% of pollen–allergic patients have IgE antibodies which cross–react with the two–EF–hand allergens in tree, grass and weed pollens. The aim of the present study was to localize Bet v 4, the two–EF–hand allergen from birch, in mature, dry pollen and to study the release of this allergen after hydration of the pollen by immunogold electron microscopy. Methods: Using completely anhydrous fixation techniques in combination with immunogold electron microscopy, we localized Bet v 4 and, for control purposes, the major birch pollen allergen Bet v 1, in dry birch pollen as well as in pollen grains after different periods of hydration. Parallel with these morphological studies, we monitored the release of Bet v 4 and Bet v 1 into aqueous supernatants of hydrated birch pollen grains by immunoblotting. Results: Bet v 4 was found in the electron–dense cytosol, in particular between the vesicles and cisternae of the endoplasmic reticulum, inside mitochondria and in the vegetative as well as in the generative nucleus. Bet v 1 was localized in similar cellular compartments except for the mitochondria. After 30 s to 1 min of hydration, Bet v 4 migrated into the pollen exine and into the aqueous supernatants. Bet v 1 also moved out of the pollen grain, though not as quickly as Bet v 4. Conclusion: Bet v 4 represents an intracellular pollen protein which, following hydration of pollen grains, rapidly migrates to the pollen surface (exine) and is washed out. This behavior explains how Bet v 4, being primarily an intracellular pollen protein, becomes available to sensitize patients.

Calcium-Binding Proteins in Type I Allergy: Elicitors and Vaccines

Type I allergy is an immunologically-mediated hypersensitivity disease with complex genetic background affecting almost 25% of the population (Kay, 1997; Lockey and Bukantz, 1998). As a major feature of their disease, allergic patients produce IgE antibodies against per se mostly harmless antigens (i.e., allergens) which, after allergen-binding, can activate effector and inducer cells of the atopic immune response (Ravetch and Kinet, 1991; Beaven and Metzger, 1993; Bieber, 1996; Stingi and Maurer, 1997). Depending on the site and duration of allergen contact and the type of immune cells involved, the manifestations of Type I allergy may greatly vary (e.g., allergic rhinitis, conjunctivitis, asthma, dermatitis, gastrointestinal disease). Progress made in the field of molecular allergen characterization has revealed that calcium-binding proteins from many sources are frequent targets for IgE antibodies of allergic patients and thus can act as widely spread elicitors of Type I allergy (Valenta et al., 1998). Calcium-binding allergens from different sources share sequence and structural similarities. Therefore, patients who are cross-sensitized to calcium-binding allergens can exhibit allergic symptoms after exposure to many allergen sources. Calcium-depletion experiments indicate that IgE antibodies of sensitized individuals recognize preferentially the calcium-bound forms of the allergens whereas the apoforms are poorly recognized. This fact opens possibilities to employ genetic engineering and synthetic peptide chemistry for the production of hypoallergenic apoforms which may be used for therapeutic vaccination against Type I allergy with a reduced rate of anaphylactic side effects.