Identification of a synthetic TLR4-agonistic peptide V<sup>77</sup>-E<sup>92</sup> derived from breast-milk α<sub>S1</sub>‑casein

Abstract

Breast-milk αS1-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 αS1-casein fulfilled two of these criteria. (i) αS1-Casein required TLR4/MD2 complex as well as cofactor CD14 to induce IL-8 secretion via TLR4 and (ii) αS1-casein bound TLR4, MD2 and CD14.3 Aim of this study was to (iii) identify a synthetic amino acid sequence derived from human αS1-casein responsible for TLR4-agonistic effects. For this, we analyzed the amino acid sequence (AAS) of αS1-casein in silico. αS1‑Casein showed to be α-helical and was likely to be intrinsically disordered in the region corresponding to R16-K99 of αS1-casein. Six truncated variants of αS1-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 αS1-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 αS1-casein was neither binding TLR4+ nor inducing IL-8 secretion. Therefore, we postulated V77-E92 derived from αS1-casein as TLR4-agonist. This was confirmed by a synthetic peptide V77-E92 derived from αS1-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, αS1-casein was proofed as an agonist directly activating TLR4. This supported our postulate that αS1-casein has at least two functions, a nutritional and an immune active one. 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). Mancek-Keber, M. & Jerala, R. Postulates for validating TLR4 agonists. Eur J Immunol 45, 356-70 (2015). 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).