Breast-milk α_S1-casein is a Toll-like receptor (TLR4) agonist which induced proinflammatory cytokine secretion. Phosphorylated α_S1-casein (P- α_S1-casein) is non-agonistic.^1,2 The objective of this study was to analyze structural characteristics underlying these observations.

Recombinant α_S1-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 α_S1-casein conformation was found at a pH-range between 7.4 and 2. α_S1-Casein bound itself (K_D-value: 2 µM) formed large aggregates (between Ø 73 nm [pH7] and Ø 826.2 nm [pH2]). Using Thioflavin T assay and atomic force microscopy showed that α_S1-casein adopted fibril-like structure. P-α_S1-casein was observed in a less α‑helical conformation, not inducing IL-8 secretion. P-α_S1-casein bound itself stronger (K_D-value: 0.5 µM) than α_S1-casein and did not form fibrils.

In conclusion, TLR4-agonistic and non-agonistic conformations of α_S1-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 α_S1-casein especially in immune system development.

1. 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).
2. 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).