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

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Abstract
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.