Differential Effects of Dry vs. Wet Heating of β-Lactoglobulin on Formation of sRAGE Binding Ligands and sIgE Epitope Recognition

Abstract

The effect of glycation and aggregation of thermally processed β-lactoglobulin (BLG) on binding to sRAGE and specific immunoglobulin E (sIgE) from cow milk allergic (CMA) patients were investigated. BLG was heated under dry conditions (water activity < 0.7) and wet conditions (in phosphate buffer at pH 7.4) at low temperature (<73 °C) and high temperatures (>90 °C) in the presence or absence of the milk sugar lactose. Nε-(carboxymethyl)-l-lysine (CML) western blot and glycation staining were used to directly identify glycation structures on the protein fractions on SDS-PAGE. Western blot was used to specify sRAGE and sIgE binding fractions. sRAGE binding was highest under wet-heated BLG independent of the presence of the milk sugar lactose. Under wet heating, high-molecular-weight aggregates were most potent and did not require the presence of CML to generate sRAGE binding ligands. In the dry system, sRAGE binding was observed only in the presence of lactose. sIgE binding affinity showed large individual differences and revealed four binding profiles. Dependent on the individual, sIgE binding decreased or increased by wet heating independent of the presence of lactose. Dry heating required the presence of lactose to show increased binding to aggregates in most individuals. This study highlights an important role of heating condition-dependent protein aggregation and glycation in changing the immunogenicity and antigenicity of cow’s milk BLG.

Binding of CML-Modified as Well as Heat-Glycated β-lactoglobulin to Receptors for AGEs Is Determined by Charge and Hydrophobicity

“…However, disentangling the effect of the presence of AGEs and 3D-changes of the protein structure upon heating induced glycation was often not possible. Recent studies highlighted the importance of separating the effect of aggregation, heating-induced exposure of hydrophobic as well as β-sheet structures and the formation of AGEs in order to explore the role of AGEs in inflammation [ 27 , 28 , 29 ]. In this study, BLG was chemically modified with glyoxylic acid to induce CML and structural changes were closely monitored.…”

“…The heating conditions used to induce protein glycation as well as the MR itself can promote protein aggregation [ 37 ]. This is a crucial determinant as it has been shown before that heating- and glycation-induced aggregation of BLG results in the formation of sRAGE binding ligands [ 28 , 29 ]. Native PAGE confirmed that in this well-defined system no aggregates were formed in any of the samples ( Figure 4 ).…”

“…However, the knowledge on the particular binding sides of these receptors to MR-modified proteins is very limited because of complexity of the structural rearrangements during the heat treatment and glycation. For instance, heating that is required to form AGEs via the MR, as well as the MR itself, promote protein aggregation which was already shown to induce an interaction with sRAGE [ 28 , 29 ]. Structural analysis of BLG-CML showed that the tertiary and secondary structure of BLG is maintained upon modification.…”

“…Only a few studies looked at the role of specific AGEs while limiting 3D-folding modifications during the glycation process [ 23 , 26 ]. However, disentangling these effects is crucial, as protein aggregation, heating, and glycation induced increases of hydrophobicity and β-sheet structures have also been reported as determinants for uptake of glycated food proteins by macrophages and the binding to AGE receptors [ 27 , 28 , 29 ]. In this study, chemical modification of the milk allergen BLG was performed to obtain a specific modification of lysine to CML, which was then compared to BLG glycated with lactose below the denaturation temperature of BLG, to avoid heat-induced structural changes of the protein.…”

Peptide Release after Simulated Infant In Vitro Digestion of Dry Heated Cow’s Milk Protein and Transport of Potentially Immunoreactive Peptides across the Caco-2 Cell Monolayer

“…However, this also resulted in some limitations, as only qualitative data was presented and allergenicity as well as immunogenicity was not measured directly. Moreover, other structures that could affect immunogenicity as well as allergenicity, such as aggregated protein that might also resist in vitro digestion, have not been considered [57,58]. transport seemed to be favored for smaller peptides (up to 13 amino acids) as well as peptides modified to lactosyl lysine and glucosyl lysine.…”