Enlarging the Toolbox for Allergen Epitope Definition with an Allergen-Type Model Protein

Abstract

BackgroundBirch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens.Method Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1.ResultsWe generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation.ConclusionOur system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

IgE and allergen‐specific immunotherapy‐induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen

“…Serum of patient 15 did not have IgG 4 against Bet v 1a but was included as additional control for IgE binding assays. Serum of patient 15 was published elsewhere . If not stated otherwise, sera from blood drawings taken 36 months after start of AIT were analyzed.…”

Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae

“…T. flavum NCS (TFLNCS) was subsequently used to query opium poppy ( Papaver somniferum ) EST databases, resulting in the isolation of two isoforms (PSONCS1 and PSONCS2) both catalyzing the formation of ( S )-norcoclaurine from dopamine and 4-HPAA, and displaying 89% conserved amino acid sequence identity, but only 40% identity compared with TFLNCS8. NCS variants from T. flavum and P. somniferum also showed 30–40% identity with members of the family of pathogenesis-related 10 (PR10) protein/Bet v1 allergens9. Investigations into the reaction mechanism leading to the formation of ( S )-norcoclaurine from dopamine and 4-HPAA suggested the two-step cyclization of a putative iminium ion intermediate1011.…”

Recombinant Allergens in Structural Biology, Diagnosis, and Immunotherapy

“…Structural homologs of allergens can also be used as scaffolds for epitope grafting, whereby the individual surface areas of allergens are transplanted onto low or nonallergenic members of the same protein (super)family. Such chimeric molecules were produced by grafting surface areas of Bet v 1 onto either a structurally related protein from celery [26] or meadow rue [27]. The norcoclaurine synthase from meadow rue was further used as a scaffold for presenting conformational epitopes of the soybean allergen Gly m 4, in an effort to characterize its IgE epitope profile [28].…”