Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS


Since its discovery over two decades ago, it has become clear that modification of protein serines/threonines by an O-linked N-acetylglucosamine (O-GlcNAc) is an essential, abundant and dynamic post-translational process in metazoa. O-GlcNAcylated proteins have been detected in both the nucleus and cytoplasm and the levels of O-GlcNAcylation respond to nutrient levels and cellular stress. O-GlcNAc has now been detected on hundreds of proteins, many of which play key roles in cellular processes. Evidence exists for the association of O-GlcNAc and neurodegenerative diseases, cancer and diabetes. Dynamic protein O-GlcNAcylation is achieved by interplay of two essential enzymes -O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), with functional similarities to the protein kinases and phosphatases, respectively. Until quite recently it was thought that protein glycosylation was restricted to eukaryotes. However, a number of landmark studies have recently uncovered that bacteria, in particular pathogenic species, possess various mechanisms for N/O-glycosylation of a range of proteins. Interestingly, a few recent reports have suggested that O-GlcNAc may also occur in bacteria. However, there are so far no examples of bacteria that reversibly O-GlcNAcylate their cytosolic proteins with the help of OGA/OGT-like proteins. If found, such bacteria could serve as ideal model organisms for the study of OGA/OGT knockouts/knockins and as testbeds for inhibitors and selectivity studies. It is my aim, building on preliminary data, to discover and characterise examples of bacterial O-GlcNAc. There are a number of examples of pathogenic bacteria subverting host immune responses by targeted interference with relevant signal transduction pathways of the host. Interestingly, a few recently described secreted bacterial glycosyltransferases also appear to modify host proteins to shut down key responses/cellular processes. Analysis of the sequence databases, reveals that there are several bacterial entries representing hOGA and hOGT apparent orthologues. Intriguingly, many of these occur in pathogenic bacteria that either have the ability to penetrate and replicate in host cells, or inject virulence factors through specific secretion systems. This projects aimed to experimentally test the hypothesis that some of these apparent OGA/OGT orthologues are secreted bacterial virulence factors, targeting specific proteins in the host. Since the beginning of the project, we have used bioinformatics to search for bacteria that contain both an O-GlcNAcase (OGA) and an O-GlcNAc transferase (OGT), based on specific search parameters. We have identified eight bacteria that contain both enzymes based on sequence database searches with the human orthologue. We have selected one of these for detailed study. We have cloned and purified both the putative OGA and OGT from this bacteria and currently characterizing these proteins kinetically and structurally. We have cultured the thermophile and have used collision induced disassociation (CID) mass spectrometry and western blotting approaches to show that this bacterium possess O-GlcNAc modified intracellular proteins. Preliminary CID mass spectrometry data has revealed 18 candidate O-GlcNAc modified proteins from the bacteria. Some of these modified proteins share similarity with signal transduction proteins. We have cloned and expressed 5 of these proteins for in vitro O-GlcNAc modification and removal assays. Beyond the end of this project, this information from these studies will help other researchers to increase the understanding of O-GlcNAc modification in eukaryotic species.

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Scientific Research
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