Vig r 6, the cytokinin-specific binding protein from mung bean (Vigna radiata) sprouts, cross-reacts with Bet v 1-related allergens and binds IgE from birch pollen allergic patients' sera

Abstract

Scope: Birch pollen associated allergy to mung bean sprouts is caused by cross-reactivity between the birch pollen allergen Bet v 1 and the mung bean allergen Vig r 1. We aimed to determine the allergenicity of the cytokinin-specific binding protein from mung bean (Vig r 6), another allergen related to Bet v 1 with only 31% sequence identity.

Methods and results: Bet v 1, Gly m 4, Vig r 1, and Vig r 6 were produced in Escherichia coli. In an ELISA, 73 and 32% of Bet v 1-sensitized birch-allergic patients' sera (n = 60) showed IgE binding to Vig r 1 and Vig r 6, respectively. Of 19 patients who reported allergic reactions or had positive prick-to-prick tests to mung bean sprouts, 79% showed IgE binding to Vig r 1 and 63% showed IgE binding to Vig r 6. Bet v 1 completely inhibited IgE binding to both mung bean allergens. Vig r 6 showed partial cross-reactivity with Vig r 1 and activated basophils sensitized with mung bean allergic patients' sera.

Conclusion: We demonstrated IgE cross-reactivity despite low sequence identity between Vig r 6 and other Bet v 1-related allergens. Thus, IgE binding to Vig r 6 may contribute to birch pollinosis-associated mung bean sprout allergy.

Keywords: Bet v 1; CSBP; Food allergy; IgE cross-reactivity; Vig r 6.

Figure 1

Sequence and structural comparison of the allergens examined in this study. (A) Sequence alignment of Bet v 1 with Bet v 1-related allergens from soybean and mung bean. (B, C) Structural comparison of Vig r 1 (B) and Vig r 6 (C), respectively, with Bet v 1. Black: residues or surface patches identical to Bet v 1; Encircled numbers: Conserved potential cross-reactive epitopes of Vig r 6; 1: p-loop; 2: surface patch centered on residue K32. The sequence alignment was generated using BioEdit 7.1.3.0 [36]. Structures were aligned and visualized using UCSF Chimera 1.6.2 [16].

Figure 2

CD spectra of purified allergens. (A) Secondary structure contents of Bet v 1.0101, Gly m 4.0101, Vig r 1.0101, and Vig r 6.0101 measured at room temperature and pH 7.4. (B) Thermal denaturation determined at 198 nm between 25 and 95°C.

Figure 3

Sensitization profiles of unselected Bet v 1-sensitized patients (Panel A) and mung bean sensitized patients (Panel B) determined by IgE ELISA.

Figure 4

Correlation of the amounts of allergen-specific IgE. IgE ELISA OD values for the different allergens were normalized to a substrate incubation period of 1 h. Negative values were set to 0.001. Spearman’s rank correlation coefficients (r values) and p values were calculated using GraphPad Prism.

Figure 5

IgE ELISA inhibition. One serum from panel A (RP) and three sera from panel B (B20, S12, and S13) were selected. Bet v 1, Vig r 1, and Vig r 6 were immobilized to microtiter plates at 10 μg/mL. Sera were preincubated with these allergens at concentrations between 20 pg/ml and 200 μg/mL and residual IgE binding to the immobilized allergens measured.

Figure 6

Basophil activation assay. Rat basophilic leukemia cells were sensitized with one serum from the Bet v 1-sensitized group (serum 4) and with three sera from the panel of mung bean sprout sensitized patients (S2, S4, and S9), and then incubated with Bet v 1, Vig r 1, and Vig r 6. Activation was determined by measuring β-hexosaminidase activity in the supernatants.