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Allergen Valency, Dose, and Fc?RI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens.

ABSTRACT: Ag-mediated crosslinking of IgE-Fc?RI complexes activates mast cells and basophils, initiating the allergic response. Of 34 donors recruited having self-reported shrimp allergy, only 35% had significant levels of shrimp-specific IgE in serum and measurable basophil secretory responses to rPen a 1 (shrimp tropomyosin). We report that degranulation is linked to the number of Fc?RI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using clustered regularly interspaced palindromic repeat-based gene editing, human RBLr?KO cells were created that exclusively express the human Fc?RI? subunit. Pen a 1-specific IgE was affinity purified from shrimp-positive plasma. Cells primed with a range of Pen a 1-specific IgE and challenged with Pen a 1 showed a bell-shaped dose response for secretion, with optimal Pen a 1 doses of 0.1-10 ng/ml. Mathematical modeling provided estimates of receptor aggregation kinetics based on Fc?RI occupancy with IgE and allergen dose. Maximal degranulation was elicited when ?2700 IgE-Fc?RI complexes were occupied with specific IgE and challenged with Pen a 1 (IgE epitope valency of ?8), although measurable responses were achieved when only a few hundred Fc?RI were occupied. Prolonged periods of pepsin-mediated Pen a 1 proteolysis, which simulates gastric digestion, were required to diminish secretory responses. Recombinant fragments (60-79 aa), which together span the entire length of tropomyosin, were weak secretagogues. These fragments have reduced dimerization capacity, compete with intact Pen a 1 for binding to IgE-Fc?RI complexes, and represent a starting point for the design of promising hypoallergens for immunotherapy.

SUBMITTER: Mahajan A

PROVIDER: S-EPMC5263102 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Allergen Valency, Dose, and FcεRI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens.

Mahajan Avanika A Youssef Lama A LA Cleyrat Cédric C Grattan Rachel R Lucero Shayna R SR Mattison Christopher P CP Erasmus M Frank MF Jacobson Bruna B Tapia Lydia L Hlavacek William S WS Schuyler Mark M Wilson Bridget S BS

Journal of immunology (Baltimore, Md. : 1950) 20161230 3

Ag-mediated crosslinking of IgE-FcεRI complexes activates mast cells and basophils, initiating the allergic response. Of 34 donors recruited having self-reported shrimp allergy, only 35% had significant levels of shrimp-specific IgE in serum and measurable basophil secretory responses to rPen a 1 (shrimp tropomyosin). We report that degranulation is linked to the number of FcεRI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using clustered regularly interspaced ...[more]

PMID: 28039304

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Project description:BackgroundPen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13.Methodology/principal findingsSerum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A(148)-E(166)) and S22 (A(243)-K(274)) were recognized by sera from 90% and 100% of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T(261)-K(274)), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity.Conclusions/significanceExperimental analyses confirmed in silico-predicted residues involved in an important antigenic region of Pen c 13. The G270A mutant of Pen c 13 has the potential to serve as an additional tool for the diagnosis/prognosis of mold allergy, and the K274A mutant, as a hypoallergenic form of the epitope, may provide a framework for the design and development of a safe and efficient therapeutic strategy for treating human allergic diseases.

Dataset Information

Allergen Valency, Dose, and Fc?RI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens.

Publications

Allergen Valency, Dose, and FcεRI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens.

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