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ObjectiveMeningococcal meningitis is a public health burden. Immunization strategies have reduced global incidence of the disease. Glycoconjugate vaccines are the most effective type of vaccine to combat most causes of meningococcal meningitis. These vaccines contain capsular polysaccharide fragments from disease-causing serogroups of Neisseria meningitidis that are chemically attached to a carrier protein. The enzymes responsible for capsular polysaccharide synthesis can serve as tools to make these critical vaccine components. One such enzyme is the N. meningitidis serogroup W capsule polymerase. This enzyme is responsible for creating the galactose-sialic acid containing capsular polysaccharide of this serogroup. Our aim in this study was to determine the binding affinities of nucleotide sugar donors CMP-sialic acid and UDP-galactose using a coupled transferase assay to inform future work to modulate polysaccharide synthesis by this enzyme.ResultsWe determined a Km of 66.8 µM for CMP-sialic acid and a Km for UDP-galactose of 3.9 µM. These values are lower than reported values for other retaining galactosyltransferases and inverting sialyltransferases respectively. There were difficulties obtaining reliable data for galactosyltransferase activity. An alternate strategy is needed to assess kinetic parameters of the separate transferase activities for this enzyme.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3596-y) contains supplementary material, which is available to authorized users.

Nucleotide‐binding oligomerization domain‐containing protein 2 (NOD2) is an intracellular innate immune receptor expressed in epithelial cells and macrophages. NOD2 is activated and stabilized upon binding the peptidoglycan fragment muramyl dipeptide (MDP). The binding process initiates an innate immune response leading to the release of proinflammatory molecules via the activation of transcription factors including NF‐kB pathway. Three Single‐nucleotide polymorphisms of NOD2 are correlated with an increased susceptibility to Crohn's disease, one of the known forms of Inflammatory bowel diseases (IBD). NOD2 CD variants are inherently unstable; however, stability can be rescued by overexpressing NOD2 interacting partner, HSP70 and through thiamet G induced increase of global O‐GlcNAc level. This study is focused on identifying O‐GlcNAc sites of NOD2, revealing other binding partners, and understanding how these processes, guide NOD2 regulation. In working towards this goal, mass spectrometry and co‐immunoprecipitation experiments were performed to identify O‐GlcNAc modification site and a novel interacting partner of NOD2 respectively. Initial data obtained demonstrated that OGT is a novel binding partner of NOD2, in complex with HSP70 and guides OGT to the NOD2 substrate. Interestingly, the data suggest the complex is changed upon NOD2 engagement with peptidoglycan fragment, MDP. This highlights that there are multiple control mechanisms involved in NOD2 regulation. Future work will investigate the position of OGT and HSP70 binding on NOD2.

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In order to distinguish infectious pathogens from the trillions of commensal bacteria on the body, humans utilize their innate immune systems, which deploy pattern recognition receptors (PRRs) to detect microbe associated molecular patterns (MAMPs) specific to certain microorganisms. The PRRs Nod1 and Nod2 detect MAMPs found in the peptidoglycan (PG) of bacterial cell walls, and upon binding initiate an immune response via the NF‐kB and other pathways, leading to the expression of pro‐inflammatory cytokines and chemokines. Misregulation of Nod1 signaling is associated with various diseases, including gastric ulcers, stomach cancer, and lung cancer. Improper Nod2 signaling has been linked to Crohn's disease, and three different Crohn's associated variants of Nod2 lead to increased susceptibility to the disease and are unstable relative to the wild type. The enzyme O‐GlcNAc Transferase (OGT), which adds a GlcNAc monosaccharide to certain serines/threonines, post‐translationally modifies both Nod1 and Nod2. This post‐translational modification (PTM) is common among thousands of proteins in the nucleus, membrane, and cytosol, and is mediated by only two enzymes: OGT, which adds GlcNAc, and O‐GlcNAcase (OGA), which removes it. The effect of this PTM is diverse among the numerous client proteins. Targets include transcription factors, including oncogenes and tumor suppressors, as well as insulin receptors, kinases, chaperones, and others. We investigate the role of O‐GlcNAcylation on Nod1 and Nod2. Modification increases the stability of both PRRs according to multiple assays, including a cellular thermal shift assay (CETSA) that measures thermal stability and a cycloheximide‐chase assay that measures cellular proteosomal degradation rates. O‐GlcNAc modification also leads to increased PG‐induced NF‐kB activity. The Crohn's associated variants of Nod2 are modified by OGT as well, and the modification rescues the particularly unstable 1007fs variant to near‐wild type. Additionally, we measure the stability effects gained from PG fragment binding and chaperone proteins.Support or Funding InformationMITZUTANI GLYCOSCIENCE FOUNDATIONNATIONAL SCIENCE FOUNDATION (CAREER CHE 1554967)NIH NATIONAL HEART, LUNG, AND BLOOD INSTITUTE (P01HL107153 to NEZ)NIH NIGMS COBRE P20GM104316‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.