A Recombinant ω-Gliadin-like D-Type Glutenin and an α-Gliadin from Wheat (Triticum aestivum): Two Immunoglobulin E Binding Proteins, Useful for the Diagnosis of Wheat-Dependent Allergies

Abstract

Among the wheat prolamins, D-type glutenins display a highly repetitive sequence similar to ω-gliadins, but they contain a cysteine, that allows them to be included in the gluten macropolymers. An ω-gliadin-like D-type glutenin, an α-gliadin, and an ω5-gliadin-like D-type glutenin were obtained as recombinant proteins and compared using synchrotron radiation circular dichroism. This technique evidenced the strong thermostability of the ω5-gliadin-like protein. The IgE reactivity of recombinant proteins was evaluated using 45 sera from wheat-allergic patients. The sera from patients diagnosed with cutaneous hypersensitivity to hydrolyzed wheat proteins often reacted with the ω-gliadin-like D-type glutenin and α-gliadin, whereas the IgE reaction was less frequent after dietary sensitization. So, these two proteins could be useful to diagnose these diseases. The sera from patients with exercise-induced anaphylaxis recognized the ω5-gliadin-like protein as a positive control and, less frequently, the other proteins tested. Only some sera from patients with baker's asthma reacted with the proteins tested.

Structural Basis of IgE Binding to α- and γ-Gliadins: Contribution of Disulfide Bonds and Repetitive and Nonrepetitive Domains

“…These recombinant proteins displayed higher contents in α-helical secondary structure (52% for rAG and 36% for rGG) compared with the natural fractions (36% for nAG and 25% for nGG). The results from SRCD are in accordance with the published data indicating that nAG contains primarily α-helical structure, 8,9,29 whereas nGG contains large amounts of α-helical and turn structures. 10,40 The recombinant α-gliadin rAG showed reduced IgE binding compared with natural α-gliadin nAG, but 90% of the sera were still reactive.…”

“…rAG has been previously cloned. 29 The same procedure was used for cloning recombinant γ-gliadin. Briefly, total RNA was extracted from immature (14 days before anthesis) wheat grains (Neepawa cultivar).…”

“…Fragments were cloned with restriction enzymes into a pET28b (Novagen) plasmid in frame with a sequence encoding the hexahistidine Tag (His-Tag). The sequences of the specific primers used in the cloning procedure are described in Supporting Information Table S.1 and in ref 29.…”

“…To overcome these problems, recombinant proteins provide an alternative method. Some recombinant prolamin allergens have already been produced and characterized. ,,− Well-defined protein fragments can also be produced by heterologous expression, and such recombinant fragments have been valuable for studying the antigenicity of LMW glutenin subunits and their two domains as well as fragments of high-molecular-weight (HMW) glutenins …”

Phosphorylation and Enzymatic Hydrolysis with Alcalase and Papain Effectively Reduce Allergic Reactions to Gliadins in Normal Mice

“…There are many allergens that have been found in wheat flour. Different types of wheat proteins cause different allergy symptoms, such as asthma and rhinitis, known as immunoglobulin E mediated inhalation of flour allergy . Among these allergies, gliadins are the major types of proteins responsible for wheat allergies in gluten .…”