Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure

Abstract

Birch pollinosis is often accompanied by adverse reactions to food due to pollen-allergen specific IgE cross-reacting with homologous food allergens. The tertiary structure of Pru av 1, the major cherry (Prunus avium) allergen, for example, is nearly identical with Bet v 1, the major birch (Betula verrucosa) pollen allergen. In order to define cross-reactive IgE epitopes, we generated and analysed mutants of Pru av 1 and Api g 1.0101, the major celery (Apium graveolens) allergen, by immunoblotting, EAST (enzyme allergosorbent test), CD and NMR spectroscopy. The mutation of Glu 45 to Trp 45 in the P-loop region, a known IgE epitope of Bet v 1, significantly reduced IgE binding to Pru av 1 in a subgroup of cherry-allergic patients. The backbone conformation of Pru av 1 wild-type is conserved in the three-dimensional structure of Pru av 1 Trp 45 , demonstrating that the side chain of Glu 45 is involved in a cross-reactive IgE epitope. Accordingly, for a subgroup of celery-allergic patients, IgE binding to the homologous celery allergen Api g 1.0101 was enhanced by the mutation of Lys 44 to Glu. The almost complete loss of IgE reactivity to the Pru av 1 Pro 112 mutant is due to disruption of its tertiary structure. Neither the mutation Ala 112 nor deletion of the Cterminal residues 155-159 influenced IgE binding to Pru av 1. In conclusion, the structure of the P-loop partially explains the crossreactivity pattern, and modulation of IgE-binding by site-directed mutagenesis is a promising approach to develop hypo-allergenic variants for patient-tailored specific immunotherapy.

Potential, pitfalls, and prospects of food allergy diagnostics with recombinant allergens or synthetic sequential epitopes

“…[10], [120], [121], [122] and [123] [124], [125], [126], [127], [128], [129] and [130] However, other studies at different shifting offsets, with different peptides sizes, or both revealed different sets of immunodominant sequential epitopes for the same allergens. [29], [124], [125], [126], [127], [130] and [131] Nevertheless, approaches to use these alleged immunodominant peptides for the risk assessment of life-threatening symptoms, as well as the prediction of persistence in food hypersensitivity, were apparently successful.…”

Correlating IgE reactivity with three-dimensional structure

“…Such substitutions may markedly affect the protein outer surface or directly involve contact residues important for the antigen-antibody interaction, thus reducing or abolishing antibody reactivity. The work presented in this issue of the Biochemical Journal by Neudecker and co-workers [6] clearly shows that the introduction of a proline residue at position 112 of Pru av 1, the major allergen of cherry, results in the disruption of the native tertiary structure of the molecule and thus an almost complete loss of IgE reactivity. Since the whole surface of a molecule is antigenic during a polyclonal response resulting from natural exposure [7], the single point mutation Ser 112 → Pro disrupting the native tertiary structure of Pru av 1 has a dramatic effect on the allergenicity of the whole protein.…”

“…We will only reach this goal if we are able to produce vaccines able to elicit strong protective immune responses independent of the patient-specific IgEbinding characteristics. As demonstrated by Neudecker et al [6], in the single case of an allergen from the pathogenesisrelated proteins, hypo-allergenic variants can be more easily and cost-efficiently produced by irreversibly preventing the folding process of an allergen, than by time-consuming mutation of residues that are assumed to be involved in the formation of IgE-binding epitopes. Such unfolded proteins, easily produced by single mutations introduced based on structural considerations, will retain almost, if not all, T-cell epitopes needed to provide B-cells with the necessary help to mount protective responses against allergens, and there is no reason to assume that this simple and efficient approach should not work in clinical practice.…”

Investigation of Mal d 1 Allelic Variants and Phylogenetic Diversity in Contemporary and Historical Polish Apple Cultivars

“…The tertiary structure of Bet v 1 contains three surface patches that have conserved-amino acid sequences and structures with proteins from a variety of food including cherry, apple, hazelnut, peach, carrot, celery, and soya (Vieths et al, 2002). Among them conserved-IgE cross reactive region is the P-loop located between b-strands 2 and 3 of Bet v 1 and its homologs (Mirza et al, 2000;Neudecker et al, 2003). Elicitation of clinical allergy symptoms appears to be dependent on IgE binding to these structures, which is in direct contrast to that observed for other food allergens with linear IgE binding epitopes such as plant allergens from peanut (Ara h 1, Ara h 2, Ara h 3), soya (G2 Glycinin, P34/Gly m Bd 30K), wheat (ω-5 Gliadin), pollen (Jun a 1, Par j 1, Par j 2), nuts (Jug r 1, Ana o 1), and animal allergens from egg (Ovalbumin, Ovomucoid), shrimp (Tropomyosin), and milk (αs2-Casein) (Bannon & Ogawa, 2006).…”