Abstract
Polcalcins are important respiratory panallergens, whose IgE-binding capacity depends on the presence of calcium. Since specific immunotherapy is not yet available for the treatment of polcalcin-sensitized patients, we aimed to develop a molecule for efficient and safe immunotherapy. We generated a hypoallergenic variant of the grass pollen polcalcin Phl p 7 by introducing specific point mutations into the allergen’s calcium-binding regions. We thereby followed a mutation strategy that had previously resulted in a hypoallergenic mutant of a calcium-binding food allergen, the major fish allergen parvalbumin. Dot blot assays performed with sera from Phl p 7-sensitized patients showed a drastically reduced IgE reactivity of the Phl p 7 mutant in comparison to wildtype Phl p 7, and basophil activation assays indicated a significantly reduced allergenic activity. Rabbit IgG directed against mutant rPhl p 7 blocked patients’ IgE binding to wildtype Phl p 7, indicating the mutant’s potential applicability for immunotherapy. Mass spectrometry and circular dichroism experiments showed that the mutant had lost the calcium-binding capacity, but still represented a folded protein. In silico analyses revealed that the hypoallergenicity might be due to fewer negative charges on the molecule’s surface and an increased molecular flexibility. We thus generated a hypoallergenic Phl p 7 variant that could be used for immunotherapy of polcalcin-sensitized individuals.
Highlights
Allergic reactions caused by pollen are a major burden for sensitized individuals
It has been demonstrated that the calcium-depleted forms of these proteins, the so called apoforms, display significantly reduced IgE reactivity as compared to the calcium-bound forms, indicating that the presence of calcium effects the allergens’ IgE-binding capacity[7,8]. It has been shown for the major fish allergen parvalbumin that mutation of the first and third amino acids of the two calcium-binding domains, which are known to be involved in calcium-coordination, can successfully lead to hypoallergenic molecules[9,10]
Our data indicate that amino acids in the same positions of the calcium-binding regions as in parvalbumins are involved in the formation of IgE-binding epitopes in case of polcalcins and that mutation of these amino acids leads to the generation of hypoallergenic polcalcins, which could be used as novel therapeutic agents for immunotherapy of polcalcin-sensitized patients
Summary
Allergic reactions caused by pollen are a major burden for sensitized individuals. Avoidance of the allergen source is impossible during the flowering period of the plants while symptomatic treatment can only reduce symptoms. One strategy to lower the risk of side effects is the genetic engineering of recombinant hypoallergenic allergen derivatives with reduced IgE-binding capacity, but retained T cell reactivity[4] For this the knowledge of the amino acids involved in the formation of the IgE-binding epitopes is crucial. It has been demonstrated that the calcium-depleted forms of these proteins, the so called apoforms, display significantly reduced IgE reactivity as compared to the calcium-bound forms (holoforms), indicating that the presence of calcium effects the allergens’ IgE-binding capacity[7,8] It has been shown for the major fish allergen parvalbumin that mutation of the first and third amino acids of the two calcium-binding domains, which are known to be involved in calcium-coordination, can successfully lead to hypoallergenic molecules[9,10]. Our data indicate that amino acids in the same positions of the calcium-binding regions as in parvalbumins are involved in the formation of IgE-binding epitopes in case of polcalcins and that mutation of these amino acids leads to the generation of hypoallergenic polcalcins, which could be used as novel therapeutic agents for immunotherapy of polcalcin-sensitized patients
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