Effects of methylglyoxal on shrimp tropomyosin structure and allergenicity during thermal processing

Abstract

This study aimed to analyze the effect of methylglyoxal (MGO) on the structure and allergenicity of shrimp tropomyosin (TM) during thermal processing. The structural changes were determined by SDS-PAGE, intrinsic fluorescence, circular dichroism, and HPLC-MS/MS. The allergenicity was evaluated by in vitro and in vivo experiments. MGO could cause conformational structural changes in TM during thermal processing. Moreover, the Lys, Arg, Asp, and Gln residues of TM were modified by MGO, which could destroy and/or mask TM epitopes. In addition, TM-MGO samples could lead to lower mediators and cytokines released from RBL-2H3 cells. In vivo, TM-MGO caused a significant reduction in antibodies, histamine, and mast cell protease 1 levels in sera. These results indicate that MGO can modify the allergic epitopes and reduce the allergenicity of shrimp TM during thermal processing. The study will help to understand the changes in the allergenic properties of shrimp products during thermal processing.

Keywords: Allergenicity; Methylglyoxal; Shrimp; Tropomyosin.

Fig. 1

(A) SDS-PAGE of shrimp TM modified by MGO during thermal processing (a: Incubated at 60, b: Incubated at 80℃, c: Incubated at 100℃, lane M: protein marker; lane 0: control; lane 1: n_TM: n_MGO = 1:4; lane 2: n_TM: n_MGO = 1:8; lane 3: n_TM: n_MGO = 1:16). (B) The IgE binding capacity of shrimp TM modified by MGO (a: control; b: n_TM: n_MGO = 1:4; c: n_TM: n_MGO = 1:8; d: n_TM: n_MGO = 1:16. The data represent the mean ± SD of triplicate measurements. ∗, p < 0.05; ∗∗, p < 0.01.).

Fig. 2

(A) UV absorption spectra, (B) Intrinsic fluorescence spectra, and (C) Circular dichroism of shrimp TM modified by MGO during thermal processing (a: Incubated at 60, b: Incubated at 80℃, c: Incubated at 100℃).

Fig. 3

(A) HPLC-MS/MS spectrum for a peptide containing Gln from TM-MGO samples. MGO adduction (*, +54) was observed on the lysine side-chain group in Arg-Arg-Ile-Gln-Leu-Leu-Glu-Glu-Asp-Leu-Glu-Arg-Ser. (B) HPLC-MS/MS spectrum for a peptide containing Asp from TM-MGO samples. MGO adduction (*, +54) was observed on the lysine side-chain group in Arg-Phe-Leu-Ala-Glu-Glu-Ala-Asp-Arg-Lys-Tyr-Asp-Glu-Val-Ala-Arg-Lys. (C) HPLC-MS/MS spectrum for a peptide containing Arg from TM-MGO samples. MGO adduction (*, +54) was observed on the lysine side-chain group in Arg-Phe-Leu-Ala-Glu-Glu-Ala-Asp-Arg-Lys-Tyr-Asp-Glu-Val-Ala-Arg-Lys. (D) HPLC-MS/MS spectrum for a peptide containing Lys from TM-MGO samples. MGO adduction (*, +54) was observed on the lysine side-chain group in Arg-Leu-Asn-Thr-Ala-Thr-Thr-Lys-Leu-Ala-Glu-Ala-Ser-Gln-Ala-Ala-Asp-Glu-Ser-Glu-Arg-Met.

Fig. 3

(A) HPLC-MS/MS spectrum for a peptide containing Gln from TM-MGO samples. MGO adduction (*, +54) was observed on the lysine side-chain group in Arg-Arg-Ile-Gln-Leu-Leu-Glu-Glu-Asp-Leu-Glu-Arg-Ser. (B) HPLC-MS/MS spectrum for a peptide containing Asp from TM-MGO samples. MGO adduction (*, +54) was observed on the lysine side-chain group in Arg-Phe-Leu-Ala-Glu-Glu-Ala-Asp-Arg-Lys-Tyr-Asp-Glu-Val-Ala-Arg-Lys. (C) HPLC-MS/MS spectrum for a peptide containing Arg from TM-MGO samples. MGO adduction (*, +54) was observed on the lysine side-chain group in Arg-Phe-Leu-Ala-Glu-Glu-Ala-Asp-Arg-Lys-Tyr-Asp-Glu-Val-Ala-Arg-Lys. (D) HPLC-MS/MS spectrum for a peptide containing Lys from TM-MGO samples. MGO adduction (*, +54) was observed on the lysine side-chain group in Arg-Leu-Asn-Thr-Ala-Thr-Thr-Lys-Leu-Ala-Glu-Ala-Ser-Gln-Ala-Ala-Asp-Glu-Ser-Glu-Arg-Met.

Fig. 4

Impact of TM-MGO on mediators release (A. β-hexosaminidase; B histamine) and cytokines release (C. IL-4; D. IL-13) from RBL-2H3 cells sensitized with anti-TM sera. a: control; b: n_TM: n_MGO = 1:4; c: n_TM: n_MGO = 1:8; d: n_TM: n_MGO = 1:16. The data represent the mean ± SD of triplicate measurements. ∗, p < 0.05; ∗∗, p < 0.01.

Fig. 5

(A) Sensitization experiments. (B) Hypersensitivity symptoms were scored on a scale from 0 (no symptoms) to 5 (death), as shown in Table S1 of the Support Information. Levels of (C) IgE, (D) IgG, (E) IgG1, (F) plasma histamine, and (G) mMCP-1 in serum from mice. Levels of (H) IL-4; (I) IL-5; (J) IL-13 and (K) IFN-γ on the secretions of cytokines from splenic lymphocytes, respectively. Results are expressed as the mean ± SD. (∗∗) p < 0.01, and (∗) p < 0.05 represent significant differences compared to the TM group.