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

Abstract

Intake of dietary advanced glycation end products (AGEs) is associated with inflammation-related health problems. Nε-carboxymethyl lysine (CML) is one of the best characterised AGEs in processed food. AGEs have been described as ligands for receptors present on antigen presenting cells. However, changes in protein secondary and tertiary structure also induce binding to AGE receptors. We aimed to discriminate the role of different protein modifications in binding to AGE receptors. Therefore, β-lactoglobulin was chemically modified with glyoxylic acid to produce CML and compared to β-lactoglobulin glycated with lactose. Secondary structure was monitored with circular dichroism, while hydrophobicity and formation of β-sheet structures was measured with ANS-assay and ThT-assay, respectively. Aggregation was monitored using native-PAGE. Binding to sRAGE, CD36, and galectin-3 was measured using inhibition ELISA. Even though no changes in secondary structure were observed in all tested samples, binding to AGE receptors increased with CML concentration of CML-modified β-lactoglobulin. The negative charge of CML was a crucial determinant for the binding of protein bound CML, while binding of glycated BLG was determined by increasing hydrophobicity. This shows that sRAGE, galectin-3, and CD36 bind to protein bound CML and points out the role of negatively charged AGEs in binding to AGE receptors.

Enhanced Uptake of Processed Bovine β‐Lactoglobulin by Antigen Presenting Cells: Identification of Receptors and Implications for Allergenicity

“…The involvement of both hydrophobicity and exposure of -sheet structures -next to or in combination with glycation of food proteins -in binding to AGE receptors has also recently been demonstrated in other studies. [20,36] Although, for non-fractionated BLG, heating seemed to be a driving force for binding to the receptors, the aggregates isolated from BLG-Glu showed enhanced binding and uptake by THP-1 cells compared to the aggregates isolated from BLG-H and BLG-Lac. This indicates that glycation of BLG, especially with glucose, also plays a role in the generation of specific binding ligands for receptors present on APCs.…”

“…The involvement of both hydrophobicity and exposure of β‐sheet structures ‐ next to or in combination with glycation of food proteins ‐ in binding to AGE receptors has also recently been demonstrated in other studies. [ 20,36 ]…”

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

“…The findings of this study suggested that diets with high AGE content can also result in higher uptake of AGE-modified peptides. As recently shown, the binding of AGE receptors depends on the concentration of food protein bound AGEs [22]. Therefore, quantitative data would be necessary to better judge the impact of the transport of AGE-modified peptides on the gastrointestinal immune system as well as the involved transport pathways.…”

“…Moreover, the generation of peptides carrying a glycation structure can modulate the inflammatory response by binding to the receptors for AGEs on antigen presenting cells [14,20]. Binding of AGEs to AGE receptors has particularly been shown for protein-bound CML and pyrroline [21,22], while for peptide bound AGEs this was only demonstrated for CML [20]. The availability of AGE-modified peptides to the gastrointestinal immune system by means of translocation across the epithelial barrier is an important determinant in the immunological response to a foreign antigen.…”

“…Therefore, transport of larger AGE-modified peptides could also contribute to the pool of dietary derived AGEs. This could be crucial as it has been shown with the example of CML that binding to AGE receptors is dependent on the concentration in which the CML is present in the vicinity of the receptors [22]. In this study, the peptide profiles of low temperature (LT) and high temperature (HT) heated MP after simulated infant in vitro digestion was compared to that of non-treated milk (NT).…”