Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy

Abstract

The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy.Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calciumbinding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE.Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.

Solution and high-pressure NMR studies of the structure, dynamics, and stability of the cross-reactive allergenic cod parvalbumin Gad m 1

“…A similar strategy was used for calcium-binding proteins from pollen. 42,43 In conclusion, our structural data show that the Ca 21 binding site and the region around V34 are important for the stabilization of the cod parvalbumin structure. Allergy-inducing IgE antibodies are directed against specific binding sites on the native conformation of the allergen.…”

“…Swoboda and colleagues recently showed that the introduction of mutations in the Ca 2+ binding sites could generate variants that do not bind IgE antibodies from fish‐allergic patients. A similar strategy was used for calcium‐binding proteins from pollen …”

Epitope specificity determines cross‐protection of a SIT‐induced IgG4 antibody

“…As described for allergic patients’ IgE antibodies, binding of mAb102.1F10 depended on the presence of protein-bound calcium. In the calcium-bound form, EF-hand allergens are supposed to expose amino acids critical for binding of antibodies on the surface of the molecule (26, 30). The binding site of mAb102.1F10 was mapped at the C-terminal portion of the molecule containing the second EF-hand.…”

“…Recombinant timothy grass pollen allergen Phl p 7 (15) was obtained from Biomay AG (Vienna, Austria). Recombinant Ole e 8 (25) from olive pollen and Bra r 1 from turnip rape pollen were expressed in Escherichia coli and purified by Nickel-affinity chromatography (26). Recombinant Che a 3 (27) from Lamb’s quarters’ pollen was expressed and purified as described (28).…”

An allergenic plant calmodulin from Artemisia pollen primes human DCs leads to Th2 polarization

“…IgE from other atopic/allergic individuals have a similar ArtCaM positive rate to Artemisia allergy patients ( p>0.1 ) Therefore, we assumed ArtCaM might work as a pan-allergen in allergic patients. Meanwhile, CBPs have been identified as highly conserved, immunological and clinical cross-reactivity allergenic proteins in several plants’ pollen ( 39 ). Previous research has reported that even though CBPs were only recognized in a few pollen-sensitized patients, they can exhibit stronger allergic reactions and polysensitization in allergic patients ( 40 , 41 ).…”