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

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 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 αS1-casein induced an IL-8 secretion of 0.95 ng/mL via TLR4. Our results indicate that αS1-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 αS1-casein, as described before, could be regulated by conformational changes induced by phosphorylation.

Structural and functional characterization of a putative de novo gene in Drosophila.

Comparative genomic studies have repeatedly shown that new protein-coding genes can emerge de novo from noncoding DNA. Still unknown is how and when the structures of encoded de novo proteins emerge and evolve. Combining biochemical, genetic and evolutionary analyses, we elucidate the function and structure of goddard, a gene which appears to have evolved de novo at least 50 million years ago within the Drosophila genus. Previous studies found that goddard is required for male fertility. Here, we show that Goddard protein localizes to elongating sperm axonemes and that in its absence, elongated spermatids fail to undergo individualization. Combining modelling, NMR and circular dichroism (CD) data, we show that Goddard protein contains a large central α-helix, but is otherwise partially disordered. We find similar results for Goddard's orthologs from divergent fly species and their reconstructed ancestral sequences. Accordingly, Goddard's structure appears to have been maintained with only minor changes over millions of years.

Root Extracts From Ononis spinosa Inhibit IL-8 Release via Interactions With Toll-Like Receptor 4 and Lipopolysaccharide.

Extracts from the roots of Ononis spinosa L. (restharrow roots) are traditionally used for the treatment of patients with urinary tract infections due to its mild diuretic activity, caused by the inhibition of renal human hyaluronidase-1 by isoflavonoids. Preliminary studies also indicated anti-inflammatory effects. The following study aimed at investigating potential anti-inflammatory effects of restharrow extracts, prepared with solvents of different polarity. A dichloromethane extract (OS1), mainly composed of isoflavonoids and triterpenes as characterized by LC-MS, showed a concentration-dependent (25-100 µg/ml) inhibition of IL-8 and TNF-α release from LPS-stimulated human neutrophils. Significant inhibition was also found for the triterpene α-onocerin and the norneolignan clitorienolactone B, isolated from OS1. Further, OS1 and both compounds significantly decreased the expression of the adhesion molecules CD11b/CD18 and conversely increased the expression of CD62L in LPS-stimulated human neutrophils. This finding corresponds to a reduced inflammatory response by the inhibition of adhesion and migration of immune cells. As all of the observed effects are potentially mediated via Toll-like receptor 4 (TLR4) signaling, TLR4 transfected HEK293 cells were incubated with OS1. LPS-induced IL-8 secretion was significantly inhibited in a concentration-dependent manner, confirming TLR4 antagonism. This inhibition, however, was in part caused by an interaction of OS1 with LPS. In addition, also an aqueous extract containing high amounts of isoflavonoid glycosides and saponins from the roots of O. spinosa showed anti-inflammatory effects by interacting with the TLR4 signaling pathway. This study rationalizes the traditional use of extracts from O. spinosa for therapy of urinary tract infections, due to its potential anti-inflammatory effects that are mediated via TLR4 receptor antagonism.

Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex.

BACKGROUND: The milk protein αS1-casein was recently reported to induce secretion of proinflammatory cytokines via Toll-like receptor 4 (TLR4). In this study, αS1-casein was identified as binder of theTLR4 ecto domain. METHODS: IL-8 secretion 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 LPS as control was monitored. Binding of αS1-casein to TLR4 was quantified by microscale thermophoresis (MST). RESULTS: αS1-casein induced secretion of IL-8 in TLR4+ cells and in MM6 cells with a six-times higher final IL-8 concentration in supernatants. IL-8 secretion was inhibited by intracellular TLR4-domain antagonist TAK-242 with an IC50-value of 259.6 nM, by ecto-domain TLR4 antagonistic mianserin with 10-51 µM and by anti-CD14-IgA. The binding constants (KD) of αS1-casein to the TLR4, MD2, and CD14 were 2.8 µM, 0.3 µM and 2.7 µM, respectively. Finally, αS1-casein showed a higher affinity to TLR4/MD2 (KD: 2.2 µM) compared to LPS (KD: 8.2 µM). CONCLUSION: Human αS1-casein induced proinflammatory effects are dependent upon binding to the TLR4 ectodomain and the presence of CD14. αS1-casein displayed stronger TLR4 agonistic activity than LPS via a different mode of action. GENERAL SIGNIFICANCE: Breast milk protein αS1-casein is a proinflammatory cytokine.

Ser71 of αS1-Casein is Phosphorylated in Breast Milk-Evidence from Targeted Mass Analysis.

SCOPE: The casein phosphoproteins in mother's milk supply calcium and phosphate ions and make them biologically available to the newborn. Human αS1-casein is of particular interest being also an autoantigen and proinflammatory cytokine. Phosphorylation of αS1-casein by casein kinase 2 completely abolishes binding to toll-like receptor 4 and proinflammatory effects. It is, however, not known, which amino acids are affected. Therefore, breast milk samples were analyzed in an effort to detect the phosphorylation sites of αS1-casein. METHODS AND RESULTS: Breast milk samples were tryptically digested. Target tandem MS analysis confirmed the known phosphorylation sites S33 and S41; evidence for pS89 was found in some samples. Experimental support for the presence of pS31 and pS34 was weak. Phosphorylation of a new site in αS1-casein, S71, was reproducibly measured in all samples, albeit at much lower intensity than pS33 and pS41. CONCLUSION: Phospho-occupancy rates varied greatly and could not be confidently correlated to other parameters within the cohort of 20 donors. The new phosphosite S71 is located in the neighborhood of the serine-rich region and may contribute to the cluster of high charge density at normal milk pH, likely exerting an influence on protein tertiary structure and thus function.

Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications.

Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2ß subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2ß interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2ß subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay.

Human casein alpha s1 induces proinflammatory cytokine expression in monocytic cells by TLR4 signaling.

SCOPE: Human casein alpha S1 (CSN1S1) is a milk-derived protein, which gives rise to sustained antibody formation after breast feeding in infancy and induces the expression of proinflammatory cytokines. So far, the cellular CSN1S1 receptor is unrecognized. Our objective was to identify the receptor employed by CSN1S1 to induce proinflammatory effects. METHODS AND RESULTS: CSN1S1 concentration and time dependently induced expression of IL-1ß, IL-8, and IL-6 in monocytic cells despite addition of polymyxin B, which completely abrogated LPS effects. Coincubation of monocytic cells with a neutralizing antibody to Toll-like receptor 4 (TLR4) but not to TLR2 inhibited CSN1S1-induced effects. In TLR4/MD2/CD14-cotransfected HEK293 cells CSN1S1 increased IL-8 expression, while untransfected cells were completely unresponsive. Furthermore, CSN1S1-induced IL1-ß secretion was impeded by inhibition of MyD88 and caspase-1. Flow cytometric in vitro assays confirmed binding of CSN1S1 to TLR4. Phosphorylation of CSN1S1 by casein-kinase 2 completely abolished proinflammatory cytokine induction and binding to TLR4. CONCLUSION: CSN1S1 is a multifunctional milk protein that exerts proinflammatory properties via TLR4 and the inflammasome pathway in a phosphorylation-dependent manner. The contribution of CSN1S1 in mother milk to the development of a potent immune system in breastfed individuals should be further assessed.

Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli.

In this work, two proteins, Z-domains and bovine casein, were auto-displayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different auto-display vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 105 and 1.55 × 105 proteins/E. coli cell, respectively. The co-auto-displayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-auto-displayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-auto-displayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-auto-displayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with auto-displayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.

Quantification of αS1-casein in breast milk using a targeted mass spectrometry-based approach.

The caseins comprise a milk protein fraction of high nutritional value and, as more recently discovered, of immunologic relevance. In particular, αS1-casein (CSN1S1) is of interest being a potential autoantigen. So far, the concentration of caseins in human milk was primarily determined by indirect methods. The aim of this study was to directly measure the CSN1S1 content in breast milk using mass spectrometry (MS). The quantification was based on tryptic CSN1S1 peptides with the best response in liquid chromatography (LC)-MS/MS analysis. Targeted experiments allowed both specific and sensitive detection at the low fmol level. For this pilot study, twenty breast milk samples of the first week post-partum were analyzed and contained between 3 and 540µg/ml CSN1S1. Limitations of CSN1S1 quantification are discussed.

Development of a surface display ELISA to detect anti-IgG antibodies against bovine αS1-casein in human sera.

The aim of the present study was to develop a surface display ELISA (SD-ELISA) for IgG-serum reaction against bovine casein αS1 (CSN1S1). In a SD-ELISA, the antigen is displayed on the surface of Escherichia coli using the autodisplay technology and whole cells of E. coli are used to coat the microplates for serum testing. After establishing the setup of the SD-ELISA with polyclonal rabbit antiserum against bovine CSN1S1, the SD-ELISA was validated with 20 human sera, of which 10 sera were proven to have an IgG-mediated reaction against bovine CSN1S1 and 10 sera were shown to be negative for this reaction. Receiver operating characteristics (ROC) analysis revealed sensitivity of 100% and a specificity of 100% at a cut-off value of 0.133. Furthermore, human serum of 48 patients with known reactivity against human CSN1S1 (31 positive and 17 negative) was examined by the newly developed SD-ELISA to exclude cross-reactivity. Twenty human sera showed an IgG-mediated reaction against bovine CSN1S1. Eleven of these sera were positive for the reactivity against human CSN1S1, and nine were negative. In conclusion it was demonstrated that the performance of SD-ELISA is comparable to established ELISA without loss in sensitivity or specificity. Based on the advantages of this method - in particular no need for time-consuming and expensive antigen production and purification - the SD-ELISA is a potent alternative to convenient methods for identification and especially high-throughput screening of new antigens in the field of food allergies.

Autoantibodies to αS1-casein are induced by breast-feeding.

BACKGROUND: The generation of antibodies is impaired in newborns due to an immature immune system and reduced exposure to pathogens due to maternally derived antibodies and placental functions. During nursing, the immune system of newborns is challenged with multiple milk-derived proteins. Amongst them, caseins are the main constituent. In particular, human αS1-casein (CSN1S1) was recently shown to possess immunomodulatory properties. We were thus interested to determine if auto-antibodies to CSN1S1 are induced by breast-feeding and may be sustained into adulthood. METHODS: 62 sera of healthy adult individuals who were (n = 37) or were not (n = 25) breast-fed against human CSN1S1 were investigated by a new SD (surface display)-ELISA. For cross-checking, these sera were tested for anti Epstein-Barr virus (EBV) antibodies by a commercial ELISA. RESULTS: IgG-antibodies were predominantly detected in individuals who had been nursed. At a cut-off value of 0.4, the SD-ELISA identified individuals with a history of having been breast-fed with a sensitivity of 80% and a specificity of 92%. Under these conditions, 35 out of 37 sera from healthy donors, who where breast-fed, reacted positively but only 5 sera of the 25 donors who were not breast-fed. The duration of breast-feeding was of no consequence to the antibody reaction as some healthy donors were only short term breast-fed (5 days minimum until 6 weeks maximum), but exhibited significant serum reaction against human CSN1S1 nonetheless. CONCLUSION: We postulate that human CSN1S1 is an autoantigen. The antigenicity is orally determined, caused by breast-feeding, and sustained into adulthood.

Development of a countergradient parking system for gradient liquid chromatography with online biochemical detection of serine protease inhibitors.

A gradient HPLC approach in combination with a countergradient system for online biochemical detection (BCD) to screen for inhibitors of serine proteases is described. For gradient separations, this novel countergradient system was developed to produce a biocompatible constant solvent composition in the BCD. The countergradient system is based on retaining complete gradients in an additional preparative HPLC column, followed by subsequent and reversible elution to the separation column effluent. Major advantages compared with existing countergradient systems are that no additional LC pumps are needed and enhanced stability. The developed countergradient system was systematically characterized applying different gradient programs. Inhibitors eluting in a postcolumn continuous flow analysis interfere with the enzymatic release of fluorescent 7-amino-4-methylcoumarin (AMC) from an AMC-labeled peptide. The inhibitory activity of eluting substances is sensitively detected as the degree of reduced fluorescence intensity. This biochemical detection system (BCD) for proteases was validated with three known inhibitors of the benzamidine type. Their IC 50 values were in good accordance with the results of conventional plate reader assays. Finally, a small library of protease inhibitors was successfully screened with the combination of the BCD and the countergradient system.