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The human milk protein αS1-casein was recently reported to induce secretion of proinflammatory cytokines via Toll-like receptor 4 (TLR4)1. In this study, the binding site of αS1-casein to TLR4 was identified by selective inhibition of the intracellular binding domain and extracellular ecto-domain of TLR4. For this, Interleukin 8 (IL-8) secretion was monitored after stimulation of TLR4/MD2 (myeloid differentiation factor 2)/CD14 (cluster of differentiation 14)-transfected HEK293 cells (TLR4+) and Mono Mac 6 cells (MM6) with recombinant αS1-casein, or lipopolysaccharide (LPS) as control. The αS1-casein-induced IL-8 secretion was inhibited by TAK-242, an antagonist of the intracellular binding site and mianserine, an antagonist of the extracellular binding domain. TAK-242 inhibited αS1-casein-induced IL-8 secretion with an IC50 of 259 nM and LPS-induced IL-8 secretion with an IC50 of 23 nM. Mianserine was found as moderate inhibitor of the αS1-casein-induced IL-8 secretion with an IC50-range between 10-51 µM. Therefore, we suggested αS1-casein as an inhibitor of the extracellular binding site of TLR4. These findings were supported by binding experiments using microscale thermophoresis (MST). Human αS1-casein bound to the purified extracellular TLR4/MD2-complex with a KD of 2.2 µM in comparison to LPS binding TLR/MD2 with a KD of 8.7 µM. Furthermore αS1-casein showed binding to MD2 with a KD of 0.3 µM and CD14 with a KD of 2.7 µM. In addition, human αS1-casein induced IL-8 secretion via TLR4 was inhibited by inhibitory anti-CD14-IgA. Human αS1-casein induced proinflammatory effects by binding to the ecto-domain of TLR4 and CD14 is required as cofactor. Hence human αS1-casein activates TLR4 in a different manner than LPS.

&lt;strong&gt;Analysis of the Binding Site of &amp;alpha;&lt;sub&gt;S1&lt;/sub&gt;-Casein to its Cellular Receptor TLR4 by Selective Inhibitors and Microscale Thermophoresis&lt;/strong&gt;