Identification of the Binding Site of Proinflammatory αS1-Casein to its Cellular Receptor-complex TLR4 Using Microscale Thermophoresis

Saenger T, Vordenbäumen S, Genich S, Nienberg C, Schulte M, Bleck E, Schneider M, Jose J

Abstract

Human αS1-casein is overexpressed in lactating mamma as well as in breast and prostatecancer and synovial tissue. Recently, we reported that human αS1-casein induces secretionof proinflammatory cytokines via Toll-like receptor 4 (TLR4) signaling in contrast to in vitrophosphorylated αS1-casein 1-4. Binding properties of breast milk protein human αS1-caseinare important for further understanding its role in formation of an immune system and rolein the inflammatory response.In this study the binding site of αS1-casein to TLR4 was identified using microscalethermophoresis (MST). MST allows the separation of bound and unbound molecules aswell as particles through a thermal gradient. A binding assay specific for the detection of theαS1-casein interactions was developed, with a self-binding constant of 2.2 μM.Moreover, clear differences in multimerization of unphosphorylated and phosphorylated αS1-casein became apparent. Using this MST assay for TLR4 interaction demonstrated thathuman αS1-casein is a stronger in vitro binding partner with a KD-value of 2.2 μM to purifiedhuman TLR4/MD2 compared to LPS with a KD-value of 8.2 μM. Additionally, human αS1-casein bound to two cofactors myeloid differentiation factor 2 (MD2) with a KD of 0.3 μMand cluster of differentiation 14 (CD14) with a KD-value of 2.7 μM. Therefore, both areinvolved in human αS1-casein recognition. Furthermore, we could support former findingsthat the unphosphorylated isoform of human αS1-casein binds to the receptor exclusively.These results emphasize the role of human αS1-casein exerting effects as competitivebinder to TLR4 in infancy nutrition and in autoimmune disease and cancer.