Phosphorylation of breast-milk αS1-casein induced conformational changes and abolished TLR4-agonisticity as well as formation of fibril structure

Breast-milk αS1-casein is a Toll-like receptor (TLR4) agonist which induced proinflammatory cytokine secretion. Phosphorylated αS1-casein (P- αS1-casein) is non-agonistic.1,2 The objective of this study was to analyze structural characteristics underlying these observations. Recombinant αS1-casein was shown to exist in two conformations, an α-helical TLR4-agonistic conformation and a non-agonistic conformation with lower α‑helical and higher random coil content. TLR4-agonstic αS1-casein conformation was found at a pH-range between 7.4 and 2. αS1-Casein bound itself (KD-value: 2 µM) formed large aggregates (between O 73 nm [pH7] and O 826.2 nm [pH2]). Using Thioflavin T assay and atomic force microscopy showed that αS1-casein adopted fibril-like structure. P-αS1-casein was observed in a less α‑helical conformation, not inducing IL-8 secretion. P-αS1-casein bound itself stronger (KD-value: 0.5 µM) than αS1-casein and did not form fibrils. In conclusion, TLR4-agonistic and non-agonistic conformations of αS1-casein could be differentiated. It was demonstrated that human caseins are able to adopt fibril structure. These kind of structures are often disease related. We postulate, that phosphorylation could be a switch of two conformations regulating immunomodulatory effects of human αS1-casein especially in immune system development. Vordenbaumen, S. et al. Human casein alpha s1 induces proinflammatory cytokine expression in monocytic cells by TLR4 signaling. Mol Nutr Food Res 60, 1079-89 (2016). Saenger, T. et al. Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex. Biochim Biophys Acta Gen Subj 1863, 632-643 (2019).