Microscale thermophoresis for measuring the interaction of the proinflammatory αS1-casein and its cellular receptor TLR4

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

Abstract

Human αS1-casein is a milk protein overexpressed in lactating mamma as well as in breast and prostate cancer and synovia tissues. Recently, we could show a lifelong immune reaction to αS1-casein depending on breastfeeding1. Furthermore, human αS1-casein is inducing secretion of proinflammatory cytokines via Toll-like receptor (TLR4) signaling2,3,4. Aim of this study was to identify a binding site of human αS1-casein to TLR4 with cofactors myeloid differentiation factor 2 (MD2) and cluster of differentiation 14 (CD14). Therefore, a microscale thermophoresis (MST) binding assay for human αS1-casein was developed, considering postulated protein properties. This binding assay was specific for the detection of the human αS1-casein interactions (KD = 2.2 µM). Moreover, clear differences in multimerization of unphosphorylated and phosphorylated αS1-casein became apperent. Using this microscale thermophoresis assay for the investigation of the TLR4 interaction demonstrated that human αS1-casein is a stronger in vitro binding partner (KD = 2.2µM) to purified human TLR4/MD2 compared to LPS (KD = 8.2µM). Additionally, both cofactors MD2 and CD14 bound to human αS1-casein (KD(MD2) = 0.3µM and KD(CD14) = 2.7µM). Therefore, both are involved in human αS1-casein recognition. Furthermore, we could support former findings that the unphosphorylated isoform of human αS1-casein binds to the receptor exclusively. These results emphasizes the role of human αS1-casein exerting effects as competitive binder to TLR4 in infancy nutrition as exogen protein and as endogen in autoimmune disease and cancer.