Physical interaction between the strawberry allergen Fra a 1 and an associated partner FaAP: Interaction of Fra a 1 proteins and FaAP

Abstract

The strawberry fruit allergens Fra a 1.01E, Fra a 1.02 and Fra a 1.03 belong to the group of pathogenesis-related 10 (PR-10) proteins and are homologs of the major birch pollen Bet v 1 and apple allergen Mal d 1. Bet v 1 related proteins are the most extensively studied allergens but their physiological function in planta remains elusive. Since Mal d 1-Associated Protein has been previously identified as interaction partner of Mal d 1 we studied the binding of the orthologous Fra a 1-Associated Protein (FaAP) to Fra a 1.01E/1.02/1.03. As the C-terminal sequence of FaAP showed strong auto-activation activity in yeast 2-hybrid analysis a novel time resolved DNA-switching system was successfully applied. Fra a 1.01E, Fra a 1.02, and Fra a 1.03 bind to FaAP with K of 4.5 ± 1.1, 15 ± 3, and 11 ± 2 nM, respectively. Fra a 1.01E forms a dimer, whereas Fra a 1.02 and Fra a 1.03 bind as monomer. The results imply that PR-10 proteins might be integrated into a protein-interaction network and FaAP binding appears to be essential for the physiological function of the Fra a 1 proteins.

PR10/Bet v1‐like Proteins as Novel Contributors to Plant Biochemical Diversity

“…[61] Although the physiological significance is not clear, Fra a proteins were further shown to form a tight binding interaction with a small protein of unknown function termed Fra an associated protein (FaAP). [40] Two models of PR10/Bet v1-like protein involvement in flavonoid biosynthesis consistent with the aforementioned lines of evidence have been proposed ( Figure 4). The first suggests that PR10/Bet v1-like proteins might function as "chemical chaperones" which bind flavonoid intermediates and shuttle them within the cell.…”

“…Shortly thereafter, isothermal titration calorimetry and crystallographic analyses confirmed that Fra a proteins differentially and selectively bind flavonoids but not early phenylpropanoid pathway intermediates (Section 3.5) [61] . Although the physiological significance is not clear, Fra a proteins were further shown to form a tight binding interaction with a small protein of unknown function termed Fra an associated protein (FaAP) [40] …”

“…metabolons) where their binding activities limit diffusion of toxic or unstable intermediates and expedite transfer from one enzyme active site to the next. The potential of PR10/Bet v1‐like proteins to form protein:protein interactions such as those in metabolons is demonstrated by the tight binding between Fra a and FaAP proteins [40] . Alternatively, the putative PR10/Bet v1‐like shuttle function might be more relevant for transport between subcellular compartments or across biological membranes.…”

“…PR10/Bet v1‐like proteins interact with other biological ligands, including proteins, [39,40] fatty acids, [25] phenolics including flavonoids, [41] tryptamines such as melatonin [42] and several classes of alkaloids [43–48] . In some cases, these interactions are proposed to modulate biochemical pathways.…”

Effect of the Strawberry Genotype, Cultivation and Processing on the Fra a 1 Allergen Content

“…The plasmid construct pQE70 fra a 1.02 generated in a previous study [ 16 , 22 ] was used for transformation in the expression strain Escherichia coli ( E . coli ) BL21 (DE3) pLysS.…”