The thermodynamically less stable form of galactose-galactofuranose (Galf)-is essential for the viability of several pathogenic species of bacteria and protozoa but absent in this form in mammals, so the biochemical pathways by which Galf-containing glycans are assembled and catabolysed are attractive sites for drug action. This potential has led to increasing interest in the synthesis of molecules containing Galf residues, their subsequent use in studies directed towards understanding the enzymes that process these residues and the identification of potential inhibitors of these pathways. Major achievements of the past several years have included an in-depth understanding of the mechanism of UDP-galactopyranose mutase (UGM), the enzyme that produces UDP-Galf, which is the donor species for galactofuranosyltransferases. A number of methods for the synthesis of galactofuranosides have also been developed, and practitioners in the field now have many options for the initiation of a synthesis of glycoconjugates containing either alpha- or beta-Galf residues. UDP-Galf has also been prepared by a number of approaches, and it appears that a chemoenzymatic approach is currently the most viable method for producing multi-milligram amounts of this important intermediate. Recent advances both in the understanding of the mechanism of UGM and in the synthesis of galactofuranose and its derivatives are highlighted in this review.
SUMMARY Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8+ T cell immunity and promoted CD4+Foxp3+ Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN+ suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.
The major structural component of the cell wall of Mycobacterium tuberculosis is a lipidated polysaccharide, the mycoyl-arabinogalactan-peptidoglycan (mAGP) complex. This glycoconjugate plays a key role in the survival of the organism, and thus, enzymes involved in its biosynthesis have attracted attention as sites for drug action. At the core of the mAGP is a galactan composed of D-galactofuranose residues attached via alternating beta-(1-->5) and beta-(1-->6) linkages. A single enzyme, glfT, has been shown to synthesize both glycosidic linkages. We report here the first high-level expression and purification of glfT by expression of the Rv3808c gene in Escherichia coli C41(DE3). Following a three-step purification procedure, 3-7 mg of protein of >95% purity was isolated from each liter of culture. We subsequently probed the substrate specificity of glfT by evaluating a panel of potential mono- and oligosaccharide substrates and demonstrated, for the first time, that trisaccharides are better substrates than disaccharides and that one disaccharide, in which the terminal D-galactofuranose residue is replaced with an L-arabinofuranose moiety, is a weak substrate. Kinetic characterization of the enzyme using four of the oligosaccharide acceptors gave K(m) values ranging from 204 microM to 1.7 mM. Through the use of NMR spectroscopy and mass spectrometry, we demonstrated that this recombinant enzyme, like the wild-type protein, is bifunctional and can synthesize both beta-(1-->6) and beta-(1-->5)-linkages in an alternating fashion. Access to purified glfT is expected to facilitate the development of high-throughput assays for the identification of inhibitors of the enzyme, which are potential antituberculosis agents.
The final step in the enzymatic synthesis of the ABO(H) blood group A and B antigens is catalyzed by two closely related glycosyltransferases, an ␣-(133)-N-acetylgalactosaminyltransferase (GTA) and an ␣-(133)-galactosyltransferase (GTB). Of their 354 amino acid residues, GTA and GTB differ by only four "critical" residues. High resolution structures for GTB and the GTA/GTB chimeric enzymes GTB/G176R and GTB/G176R/ G235S bound to a Glycosyltransferases synthesize carbohydrate moieties of glycoconjugates by catalyzing the sequential addition of monosaccharides from specific donors to specific acceptors. The ubiquitous presence of glycolipids and glycoproteins in all living systems underlines the importance of the glycosyltransferases superfamily, and the DNA of all domains of life encode for a large number of these enzymes (1). To date, crystal structures of glycosyltransferases have displayed a high degree of structural similarity even when there is low sequence homology (2-4). As such, glycosyltransferases provide an excellent example of the preferential conservation of structural phenotype over the conservation of sequence identity (2), which indicates that the mechanism of glycosylation, although not yet fully understood, has been conserved.
The 126 possible conformations of 1,2,3-propanetriol (glycerol) have been studied by ab initio molecular orbital and density functional theory calculations in the gas and aqueous phases at multiple levels of theory and basis sets. The partial potential energy surface for glycerol as well as an analysis of the conformational properties and hydrogen-bonding trends in both phases have been obtained. In the gas phase at the G2(MP2) and CBS-QB3 levels of theory, the important, low-energy conformers are structures 100 and 95. In the aqueous phase at the SM5.42/HF/6-31G* level of theory, the lowest energy conformers are structures 95 and 46. Boltzmann distributions have been determined from these high-level calculations, and good agreement is observed when these distributions are compared to the available experimental data. These calculations indicate that the enthalpic and entropic contributions to the Gibbs free energy are important for an accurate determination of the conformational and energetic preferences of glycerol. Different levels of theory and basis sets were used in order to understand the effects of nonbonded interactions (i.e., intramolecular hydrogen bonding). The efficiency of basis set and level of theory in dealing with the issue of intramolecular hydrogen bonding and reproducing the correct energetic and geometrical trends is discussed, especially with relevance to practical computational methods for larger polyhydroxylated compounds, such as oligosaccharides.
Bacterial capsules are surface layers made of long-chain polysaccharides. They are anchored to the outer membrane of many Gram-negative bacteria, including pathogens such as Escherichia coli , Neisseria meningitidis , Haemophilus influenzae , and Pasteurella multocida . Capsules protect pathogens from host defenses including complement-mediated killing and phagocytosis and therefore represent a major virulence factor. Capsular polysaccharides are synthesized by enzymes located in the inner (cytoplasmic) membrane and are then translocated to the cell surface. Whereas the enzymes that synthesize the polysaccharides have been studied in detail, the structure and biosynthesis of the anchoring elements have not been definitively resolved. Here we determine the structure of the glycolipid attached to the reducing terminus of the polysialic acid capsular polysaccharides from E. coli K1 and N. meningitidis group B and the heparosan-like capsular polysaccharide from E. coli K5. All possess the same unique glycolipid terminus consisting of a lyso-phosphatidylglycerol moiety with a β-linked poly-(3-deoxy- d - manno -oct-2-ulosonic acid) (poly-Kdo) linker attached to the reducing terminus of the capsular polysaccharide.
SummaryPathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction.
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