Deciphering Changes in the Structure and IgE-Binding Ability of Ovalbumin Glycated by α-Dicarbonyl Compounds under Simulated Heating

Abstract

As a complex reaction, biological consequences of the Maillard reaction (MR) on dietary proteins need to be deciphered. Despite previous studies on the structural and antigenic properties of ovalbumin (OVA) by MR, associated changes induced by specific MR intermediates and their downstream products are largely unknown. This study focused on the impacts of glycation by α-dicarbonyl compounds (α-DCs), intermediates of MR and precursors of advanced glycation end-products (AGEs), on the structural and IgE-binding properties of ovalbumin (OVA) under simulated heating. Methylglyoxal (MGO), glyoxal (GO), and butanedione (BU) were selected as typical α-DCs to generate glycated OVA with different AGE-modifications (AGE-Ms). The results showed that reactions between OVA and α-DCs generated OVA-AGE with various degrees of modification and conformational unfolding, and the reactivity of α-DCs followed the order GO > MGO > BU. Depending on the precursor type, the levels of 10 specific AGEs were verified, and the amounts of total AGEs increased with heating temperature and α-DC dosage. Compared to native OVA, glycated OVA showed reduced IgE-binding levels but with sRAGE-binding ligands, the extent of which was associated with the contents of total AGEs and Nε-carboxymethyllysine, and changes in certain protein conformational structures. High-resolution mass spectrometry further identified different AGE-Ms on the Lys and Arg residues of OVA, confirming variations in the glycation sites and their associations with the immunoreactive epitopes of OVA under different conditions.

Effects of Allergen‐Specific and Non‐Specific AGEs on the Allergenicity of Ovalbumin in a Mouse Model of Food Allergy

“…[33,34] In our previous report, we demonstrated that glycation of OVA with typical AGE precursors like MGO and glyoxal could reduce the IgEbinding level of OVA by up to 72% when heating at 95 °C. [20] The locations of AGE-modified sites, as revealed by high-resolution mass spectrometry (MS), were found to fall within the sequences of several known epitopes of OVA, thus providing a masking effect on the epitope-sIgE recognition. However, it is worth noting that a lower IgE-binding ability does not guarantee hypoallergenicity in vivo.…”

“…Both diets were manufactured by Xietong Bioengineering Co., Ltd. (Nanjing, China), and as shown in Table S1, Supporting Information the two diets were free of allergen and identical in calories and macronutrients level. Generation of fluorescent AGEs in the OVA-AGE or H-AGE diet was determined by fluorometry as described by the previous report, [56] and the contents of 10 specific AGEs, namely, CML, CEL, MG-H1/3, MG-H2, G-H1, pyrraline, pentosidine, argpyrimidine, and MOLD were quantified by an isotope dilution-based UHPLC-MS/MS method as indicated by Zhang et al [20] In Vitro IgE-Binding Ability of OVA-AGE: The IgE-binding ability of OVA-AGE was analyzed by an indirect competitive ELISA assay according to the protocol reported elsewhere. [20] In brief, the OVA solution (0.5 mg mL −1 ) dissolved in 0.05 M carbonate buffer solution (pH 9.6) was coated on a 96-well plate (2 μg protein per well) and then kept overnight at 4 °C.…”

“…Generation of fluorescent AGEs in the OVA-AGE or H-AGE diet was determined by fluorometry as described by the previous report, [56] and the contents of 10 specific AGEs, namely, CML, CEL, MG-H1/3, MG-H2, G-H1, pyrraline, pentosidine, argpyrimidine, and MOLD were quantified by an isotope dilution-based UHPLC-MS/MS method as indicated by Zhang et al [20] In Vitro IgE-Binding Ability of OVA-AGE: The IgE-binding ability of OVA-AGE was analyzed by an indirect competitive ELISA assay according to the protocol reported elsewhere. [20] In brief, the OVA solution (0.5 mg mL −1 ) dissolved in 0.05 M carbonate buffer solution (pH 9.6) was coated on a 96-well plate (2 μg protein per well) and then kept overnight at 4 °C. After blocking with 3% bovine serum albumin (BSA) solution, the plate was co-incubated with 100 μL of the mouse serum (1:100 diluted in PBS) collected in Animal Experimental Section (OVA group) and 100 μL of OVA-AGE solution (2 mg mL −1 ) at 37 °C for 1 h. Afterward, 100 μL of HRP goat anti-mouse IgE antibody (1:2500) was added to the plate and further incubated for 1 h at 37 °C.…”

“…Considering this, this study aimed to investigate the role of different types of AGEs on the allergenic manifestation of a model food allergen, ovalbumin (OVA) in a BALB/c mouse model. The OVA-specific AGEs were prepared by glycating OVA with a highly reactive AGE precursor, methylglyoxal (MGO) as we earlier reported, [20] and the OVA-non-specific AGEs were obtained by heating an allergen-free chow diet according to previous studies. [21,22] The allergic outcomes were measured by targeting key aspects of the immune responses in food allergy, including clinical symptom signs, specific antibody isotypes, lymphocyte subpopulation and related cytokines, injuries in the intestinal barrier, and profiling of the fecal GM.…”

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

“…Maillard reaction is induced by reducing sugars, which could alter the structures and physicochemical properties of dietary proteins due to sugar degradation products. a-Dicarbonylic compounds (a-DCs) like glyoxal (GO) and methylglyoxal (MGO) are important intermediates of the Maillard reaction that exist during thermal processing ( Dasanayaka et al, 2022 , Gupta et al, 2018 , Zhang et al, 2022 ). In addition, the a-DCs also inevitably react with the shrimp protein as the shrimp reheats.…”