Background: Phl p 4 is a major pollen allergen but exhibits lower allergenicity than other major allergens. The natural protein is glycosylated and shows cross-reactivity with related and structurally unrelated allergens.
Objective: We sought to determine the high-resolution crystal structure of Phl p 4 and to evaluate the immunologic properties of the recombinant allergen in comparison with natural Phl p 4.
Methods: Different isoallergens of Phl p 4 were expressed, and the nonglycosylated mutant was crystallized. The specific role of protein and carbohydrate epitopes for allergenicity was studied by using IgE inhibition and basophil release assays.
Results: The 3-dimensional structure was determined by using x-ray crystallography at a resolution of 1.9 Å. The allergen is a glucose dehydrogenase with a bicovalently attached flavin adenine dinucleotide. Glycosylated and nonglycosylated recombinant Phl p 4 showed identical inhibition of IgE binding, but compared with natural Phl p 4, all recombinant isoforms displayed a reduced IgE-binding inhibition. However, the recombinant protein exhibited an approximately 10-fold higher potency in basophil release assays than the natural protein.
Conclusion: The crystal structure reveals the compact globular nature of the protein, and the observed binding pocket implies the size of the natural substrate. Plant-derived cross-reactive carbohydrate determinants (CCDs) appear to reduce the allergenicity of the natural allergen, whereas the Pichia pastoris-derived glycosylation does not. Our results imply yet undescribed mechanism of how CCDs dampen the immune response, leading to a novel understanding of the role of CCDs.
Keywords: BBE; CCD; CD; Circular dichroism; Cross-reactive carbohydrate determinant; FAD; FSAS; Flavin adenine dinucleotide; Fractional solvent-accessible surfaces; GPA; Grass pollen allergen; Grass pollen allergy; Phl p 4; RMSD; Reticuline oxidase (informally known as the berberine bridge enzyme); Root-mean-square deviation; Sec c 4; basophil release assay; bicovalently attached flavin adenine dinucleotide; cross-reactive carbohydrate determinant; dehydrogenase; recombinant allergen.
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