The mechanism of reduced IgG/IgE-binding of β-lactoglobulin by pulsed electric field pretreatment combined with glycation revealed by ECD/FTICR-MS

Abstract

Bovine β-lactoglobulin (β-Lg) is a major allergen existing in milk and causes about 90% of IgE-mediated cow's milk allergies. Previous studies showed that pulsed electric field (PEF) treatment could partially unfold the protein, which may contribute to the improvement of protein glycation. In this study, the effect of PEF pretreatment combined with glycation on the IgG/IgE-binding ability and the structure of β-Lg was investigated. The result showed that PEF pretreatment combined with glycation significantly reduced the IgG and IgE binding abilities, which was attributed to the changes of secondary and tertiary structure and the increase in glycation sites and degree of substitution per peptide (DSP) value determined by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD/FTICR-MS). Unexpectedly, glycation sites (K47, K91 and K135) added by two mannose molecules were identified in glycated β-Lg with PEF pretreatment. Moreover, the results indicated that PEF pretreatment at 25 kV cm for 60 μs promoted the reduction of IgG/IgE-binding capacity by increasing the glycation degree of β-Lg, whereas single PEF treatment under the same conditions markedly enhanced the IgG/IgE-binding ability by partially unfolding the structure of β-Lg. The results suggested that ECD/FTICR-MS could help us to understand the mechanism of reduction in the IgG/IgE-binding of β-Lg by structural characterization at the molecular level. Therefore, PEF pretreatment combined with glycation may provide an alternative method for β-Lg desensitization.

Influence of moderate electric fields in β-lactoglobulin thermal unfolding and interactions

“…These conditions are consistent with the ones used in pulsed electric field (PEF) technology or considered in in silico approaches through molecular dynamic simulations (English & Waldron, 2015;Zhao, Yang, & Zhang, 2012). EF effects in whey proteins, and particularly in β-lg, have been demonstrated under PEF treatments where tertiary and secondary structure modifications were observed under voltage gradients above 12 kV/cm (Perez & Pilosof, 2004;Xiang, Ngadi, Ochoa-Martinez, & Simpson, 2011;Yang et al, 2018). Another application of EF technology is the use of EF of low intensity, relative to PEF, known as moderate electric fields (MEF).…”

“…Changes in β-lg structural features upon EF effects have been revealed by PEF treatments (at different EF magnitudes, exposure time and without synergy with temperature), exhibiting increased Trp exposure, increased ANS binding, secondary structure changes and differences in immunoglobulins binding (Xiang et al, 2011;Yang et al, 2018). After establishing the influence of EF strength and electrical frequency in the unfolding profile of β-lg and T m , additional experiments were focused in the assessment of structural changes induced by MEF at representative conditions -i.e.…”

Electric field effects on proteins – Novel perspectives on food and potential health implications

“…This implies that their effect on the allergenic profile of proteins may be distinctive. β-Lg treated by PEF suffered a partial unfolding of the structure contributing to increase the binding to immunoglobulins; however when the PEF treatment was conjugated with glycation of the protein, binding suffered a drastic reduction (Yang, Tu, Wang, Zhang, Kaltashov, et al, 2018). Low intensity PEF treatments of ovalbumin also resulted in the partial unfolding of the protein and the increased binding to antibodies.…”

Production of Liquid Milk Protein Concentrate with Antioxidant Capacity, Angiotensin Converting Enzyme Inhibitory Activity, Antibacterial Activity, and Hypoallergenic Property by Membrane Filtration and Enzymatic Modification of Proteins

“…In some cases, physical- [42][43][44] and enzymatic- [45][46][47][48] modifications of proteins have been adopted for a similar objective. Furthermore, combined physical and biochemical technologies have been adopted for the reduction of milk protein allergens [49][50][51][52][53][54][55][56]. In some cases, new epitopes (neoepitopes) or hidden epitopes may even be produced during cow milk processing due to denaturation of native allergen (cryptotopes) [57].…”