Project description:Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown, in part due to the lack of tools with which to study the glycan. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN and EndoNDM on streptavidin-coated magnetic or agarose beads. We showed that immobilizing EndoN improves enzyme usefulness by allowing for effective removal of the enzyme from samples, while retaining hydrolase activity. Additionally, immobilization of EndoNDM allowed for the enrichment of polysialylated proteins from complex mixtures for their identification via mass spectrometry. We identified QSOX2 as a novel polysialylated protein secreted from MCF-7 cells. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.
Project description:Sulforaphane is a naturally occurring, potent antioxidant and anti-inflammatory compound, found in cruciferous plants such as broccoli. Recently there have been a large number of clinical trials assessing broccoli sprout extracts as sulforaphane-based therapies for conditions including fibrosis, cancer and preeclampsia. As sulforaphane is orally administered, there is also the potential for impact on the gut microbiome. Here, we have determined the effect of sulforaphane on the growth of 43 common human gastrointestinal bacterial commensals and pathogens, which represented the four main phyla found in the human gastrointestinal microbiome. The pathogenic Escherichia coli strain ECE2348/69 showed the most significant increases in growth in the presence of sulforaphane compared to control conditions. Proteomic analysis of this isolate showed that sulforaphane increased anaerobic respiration, whilst metabolomic profiling identified differentially produced metabolites involved in amino acid biosynthesis and known to decrease inflammation in human cells. Therefore, sulforaphane can increase growth of specific gastrointestinal bacterial isolates, correlating with increased production of anti-inflammatory metabolites, that may provide a novel mechanism for modulating inflammatory states in patients.