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

Abstract

Scope: -lactoglobulin (BLG) is a major cow milk allergen encountered by the immune system of infants fed with milk-based formulas. To determine the effect of processing on immunogenicity of BLG, this article characterized how heated and glycated BLG are recognized and internalized by APCs. Also, the effect of heat-induced structural changes as well as gastrointestinal digestion on immunogenicity of BLG is evaluated. Methods and results: The binding and uptake of BLG from raw cow milk and heated either alone (BLG-H) or with lactose/glucose (BLG-Lac and BLG-Glu) to the receptors present on APCs are analyzed by ELISA and cell-binding assays. Heated and glycated BLG is internalized via galectin-3 (Gal-3)and scavenger receptors (CD36 and SR-AI) while binding to the receptor for advanced glycation end products (R AGE) does not cause internalization. Receptor affinity of BLG is dependent on increased hydrophobicity, -sheet exposure and aggregation. Digested glycated BLG maintained binding to sRAGE and Gal-3 but not to CD36 and SR-AI, and is detected on the surface of APCs. This suggests a mechanism via which digested glycated BLG may trigger innate (via RAGE) and adaptive immunity (via Gal-3). Conclusions: This study defines structural characteristics of heated and glycated BLG determining its interaction with APCs via specific receptors thus revealing enhanced immunogenicity of glycated versus heated BLG.

No Glycation Required: Interference of Casein in AGE Receptor Binding Tests

“…Therefore, the presence of casein can interfere in ELISA assays when determining binding of AGE receptors to heated MP. Next to AGEs, it has also been shown that other 3D-structural properties such as hydrophobicity and aggregation of food proteins can result in binding to AGE receptors [2,3,8]. Therefore, the role of the 3D structure of casein in binding to sRAGE was further investigated using dot blot analysis (Figure 3).…”

“…For both receptors, negative charge, aggregation, and hydrophobicity have been suggested as important determinants for their binding to ligands [3,7]. Moreover, there is evidence in literature that, e.g., sRAGE recognizes high molecular weight ligands formed upon aggregation of proteins [2,8,10]. The hydrophobic elements of the casein molecules are located in the inside of the casein micelle, whereas the negatively charged ends of the κ-casein point to the outside of the micelle [11].…”

“…There is a rising interest in including in vitro digestion experiments in the evaluation of immunogenicity and allergenicity of food proteins [24]. This has already been performed in some studies that investigated immunogenicity of MP [8,25]. As casein is hydrolyzed upon digestion, it was verified whether the interference of casein in inhibition ELISA for sRAGE and CD36 binding is still observed after in vitro digestion at different time points of digestion.…”

“…Binding to sRAGE and CD36 was measured using inhibition ELISA as described by Teodorowicz et al [8]. Briefly, MP samples before and after digestion were adjusted to 200 µg/mL protein concentration and incubated with sRAGE and CD36, respectively.…”

“…Immunogenicity and allergenicity of milk protein (MP) can be altered by heating [1]. Next to changes in hydrophobicity, negative charge, and protein aggregation, advanced glycation end products (AGEs) have been identified as a factor that modulate immunogenicity [2][3][4][5][6][7][8]. The receptors for AGEs have an important role in the recognition of AGEs by innate immune cells.…”