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Structural Analysis of Breast-Milk α_S1-Casein: An α-Helical Conformation Is Required for TLR4-Stimulation.

ABSTRACT: Breast-milk α_S1-casein is a Toll-like receptor 4 (TLR4) agonist, whereas phosphorylated α_S1-casein does not bind TLR4. The objective of this study was to analyse the structural requirements for these effects. In silico analysis of α_S1-casein indicated high α-helical content with coiled-coil characteristics. This was confirmed by CD-spectroscopy, showing the α-helical conformation to be stable between pH 2 and 7.4. After in vitro phosphorylation, the α-helical content was significantly reduced, similar to what it was after incubation at 80 °C. This conformation showed no in vitro induction of IL-8 secretion via TLR4. A synthetic peptide corresponding to V⁷⁷-E⁹² of α_S1-casein induced an IL-8 secretion of 0.95 ng/mL via TLR4. Our results indicate that α_S1-casein appears in two distinct conformations, an α-helical TLR4-agonistic and a less α-helical TLR4 non-agonistic conformation induced by phosphorylation. This is to indicate that the immunomodulatory role of α_S1-casein, as described before, could be regulated by conformational changes induced by phosphorylation.

SUBMITTER: Saenger T

PROVIDER: S-EPMC10855866 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Structural Analysis of Breast-Milk αS1-Casein: An α-Helical Conformation Is Required for TLR4-Stimulation.

Saenger Thorsten T Schulte Marten F MF Vordenbäumen Stefan S Hermann Fabian C FC Bertelsbeck Juliana J Meier Kathrin K Bleck Ellen E Schneider Matthias M Jose Joachim J

International journal of molecular sciences 20240201 3

Breast-milk αS1-casein is a Toll-like receptor 4 (TLR4) agonist, whereas phosphorylated αS1-casein does not bind TLR4. The objective of this study was to analyse the structural requirements for these effects. In silico analysis of αS1-casein indicated high α-helical content with coiled-coil characteristics. This was confirmed by CD-spectroscopy, showing the α-helical conformation to be stable between pH 2 and 7.4. After in vitro phosphorylation, the α-helical con ...[more]

PMID: 38339021

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Project description:BackgroundThe milk-derived protein human Casein alpha s1 (CSN1S1) has recently been detected in blood cells and was shown to possess proinflammatory properties. In the present study, we investigated the effect of CSN1S1 on the differentiation of monocytes.MethodsPrimary human monocytes were stimulated with recombinant CSN1S1 and compared to cells stimulated with GM-CSF/IL-4 or M-CSF/IFNγ. Morphological changes were assessed by microscopy and quantification of surface markers of differentiation by FACS analysis. Phagocytic activity of CSN1S1 stimulated cells was measured by quantification of zymosan labeled particle uptake. The role of mitogen activated protein kinases for CSN1S1-induced differentiation of monocytes and proinflammatory cytokine expression was assessed by supplementation of specific inhibitors.ResultsCSN1S1 at a concentration of 10 μg/ml resulted in morphological changes (irregular shape, pseudopodia) and aggregation of cells, comparable to changes observed in M-CSF/IFNγ differentiated macrophages. Surface marker expression was altered after 24 h with an upregulation of CD14 (mean 2.5 fold) and CD64 (1.9 fold) in CSN1S1 stimulated cells. CSN1S1 treated cells showed a characteristic surface marker pattern for macrophages after 120 h of incubation (CD14high, CD64high, CD83low, CD1alow) comparable to changes observed in M-CSF/IFNγ treated monocytes. Furthermore, phagocytic activity was increased 1.4 and 1.9 fold following stimulation with 10 μg/ml CSN1S1 after 24 and 48 h, respectively. Early GM-CSF, but not GM-CSF/IL-4 induced differentiation of monocytes towards dendritic cells (DC) was inhibited by addition of CSN1S1. Finally, CSN1S1 induced upregulation of CD14 was impeded by inhibition of ERK1/2, while inhibition of the mitogen activated protein kinases JNK and p38 did not influence cellular differentiation. However, JNK and p38 inhibitors impeded CSN1S1 induced secretion of the proinflammatory cytokines IL-1b or IL-6.ConclusionsCSN1S1 skews in vitro differentiation of monocytes towards a macrophage-like phenotype. Data is accumulating that functions of CSN1S1 are beyond nutritional properties and include immunomodulatory effects.

Dataset Information

Structural Analysis of Breast-Milk α_S1-Casein: An α-Helical Conformation Is Required for TLR4-Stimulation.

Publications

Structural Analysis of Breast-Milk α<sub>S1</sub>-Casein: An α-Helical Conformation Is Required for TLR4-Stimulation.

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