guanosine-diphosphate-mannose and mannosamine

The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. Cell-free reactions utilizing cellular membranes of mycobacteria as the enzyme source indicated that ManN inhibited the synthesis of phosphatidylinositol mannosides, early precursors to LAM. A selection strategy was devised to screen a Mycobacterium tuberculosis genomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) encoding a target of ManN would impart a ManN-resistant phenotype under these conditions. This strategy led to the identification of pimB, whose deduced amino acid sequence shows similarity to mannosyltransferases and other glycosyltransferases. Partially purified recombinant PimB protein from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of triacylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidylinositol monomannoside.

Topics: Bacterial Proteins; Drug Resistance, Microbial; Escherichia coli; Genes, Bacterial; Genomic Library; Guanosine Diphosphate Mannose; Hexosamines; Lipopolysaccharides; Mannosides; Mannosyltransferases; Molecular Sequence Data; Mycobacterium smegmatis; Mycobacterium tuberculosis; Phosphatidylinositols; Recombinant Proteins; Selection, Genetic; Spectrometry, Mass, Fast Atom Bombardment

This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum, and the presence in the infected erythrocyte of the respective precursors. In in vitro cultures, P. falciparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or O-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into O-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. falciparum, using exogenous glycosyl donors and acceptors. Our studies show that O-glycosylation is the major form of protein glycosylation in intraerythrocytic P. falciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A., Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372, 645].

Topics: Amidohydrolases; Animals; Carbohydrate Sequence; Carbon Radioisotopes; Chromatography, Gel; Erythrocytes; Ethanolamine; Ethanolamines; Fucose; Galactose; Glucosamine; Glycoproteins; Glycosylation; Guanosine Diphosphate Mannose; Hexosamines; Humans; Malaria, Falciparum; Molecular Sequence Data; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Plasmodium falciparum; Polyisoprenyl Phosphate Oligosaccharides; Proteins; Protozoan Proteins; Tritium