Breast-milk α_S1-casein was suggested as an agonist of the Toll-like receptor 4 (TLR4).¹ Pathogen recognition receptor TLR4 responds to lipopolysaccharides and a wide range of molecules, from proteins to metal ions. In consequence, three criteria are required to validate agonists which directly activate TLR4 and exclude TLR4-agonisticity through contaminants.² Recently, we demonstrated that α_S1-casein fulfilled two of these criteria. (i) α_S1-Casein required TLR4/MD2 complex as well as cofactor CD14 to induce IL-8 secretion via TLR4 and (ii) α_S1-casein bound TLR4, MD2 and CD14.³ Aim of this study was to (iii) identify a synthetic amino acid sequence derived from human α_S1-casein responsible for TLR4-agonistic effects.

For this, we analyzed the amino acid sequence (AAS) of α_S1-casein in silico. α_S1‑Casein showed to be α-helical and was likely to be intrinsically disordered in the region corresponding to R¹⁶-K⁹⁹ of α_S1-casein. Six truncated variants of α_S1-casein coding for parts of the AAS were purified from Escherichia coli. These variants were tested for binding to HEK293 cells transfected with TLR4 (TLR4⁺) by flow cytometry and their induction of IL-8 secretion via TLR4. Variants of α_S1-casein truncated at the N-terminus (E³⁵-W¹⁸⁵, R⁵⁷-W¹⁸⁵, V⁷⁷-W¹⁸⁵) bound TLR4⁺ induced lower IL-8 secretion with less AAS (7.5 ng/ml, 4.8 ng/ml, 3.6 ng/ml). Variant corresponding to E⁹³-W¹⁸⁵ of α_S1-casein was neither binding TLR4⁺ nor inducing IL-8 secretion. Therefore, we postulated V⁷⁷-E⁹² derived from α_S1-casein as TLR4-agonist. This was confirmed by a synthetic peptide V⁷⁷-E⁹² derived from α_S1-casein, which induced an IL-8 secretion of 0.95 ng/ml. Hence, the third criteria of TLR4-agonists fulfilled and activation of TLR4 through contamination was excluded.

In conclusion, α_S1-casein was proofed as an agonist directly activating TLR4. This supported our postulate that α_S1-casein has at least two functions, a nutritional and an immune active one.

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. Mancek-Keber, M. & Jerala, R. Postulates for validating TLR4 agonists. Eur J Immunol 45, 356-70 (2015).
3. 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).