Cryptosporidium parvum sporozoite pellicle antigen recognized by a neutralizing monoclonal antibody is a beta-mannosylated glycolipid.
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The protozoan parasite Cryptosporidium parvum is an important cause of diarrhea in humans, calves, and other mammals worldwide. No approved vaccines or parasite-specific drugs are currently available for the control of cryptosporidiosis. To effectively immunize against C. parvum, identification and characterization of protective antigens are required. We previously identified CPS-500, a conserved, neutralization-sensitive antigen of C. parvum sporozoites and merozoites defined by monoclonal antibody 18.44. In the present study, the biochemical characteristics and subcellular location of CPS-500 were determined. CPS-500 was chloroform extractable and eluted with acetone and methanol in silicic acid chromatography, consistent with being a polar glycolipid. Following chloroform extraction and silicic acid chromatography, CPS-500 was isolated by high-pressure liquid chromatography for glycosyl analysis, which indicated the presence of mannose and inositol. To identify which component of CPS-500 comprised the neutralization-sensitive epitope recognized by 18.44, the ability of the monoclonal antibody to bind CPS-500 treated with proteases, or with alpha- or beta-glycosidases, was determined. Monoclonal antibody 18.44 did not bind antigen treated with beta-D-mannosidase but did bind antigen treated with alpha-D-mannosidase, other alpha- or beta-glycosidases, or a panel of proteases. These data indicated that the target epitope was dependent on terminal beta-D-mannopyranosyl residues. By immunoelectron microscopy, 18.44 binding was localized to the pellicle and an intracytoplasmic tubulovesicular network in sporozoites. Monoclonal antibody 18.44 also bound to antigen deposited and released onto substrate over the course travelled by gliding sporozoites and merozoites. Surface localization, adhesion and release during locomotion, and neutralization sensitivity suggest that CPS-500 may be involved in motility and invasion processes of the infective zoite stages. (+info)
Determination of the anomeric configurations of Corbicula ceramide di- and trihexoside by chromium trioxide oxidation.
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The anomeric configurations of Corbicula ceramide dihexoside and ceramide trihexoside were determined by chromium trioxide oxidation and the structures of these lipids were shown to be Man-beta(1 leads to 4)-Glc-beta(1 leads to 1)-ceramide and Man-alpha(1 leads to 4)-Man-beta(1 leads to 4)-Glc-beta(1 leads to 1)-ceramide. These results are compatible with those obtained by enzymic hydrolysis reported previously. (+info)
Molecular recognition of concanavalin A on mannoside diacetylene lipid monolayer at the air-water interface.
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The interaction of p-10,12-pentacosadiyne-1-n-phenylamide alpha-D-mannopyranoside (MPDA) with protein concanavalin A (Con A) was studied at the air/water interface. The expansion of molecular area of PDA (10,12-pentacosadiynoic acid)/MPDA mixed monolayer after injection of Con A in subphase shows strong interaction between Con A and the monolayer. The maximum expansion of molecular area decreases as the molar ratio of MPDA increases due to the steric hindrance effect. By using enzyme mannosidase to cut-off the mannoside headgroup of MPDA, expansion of molecular area was greatly reduced, indicating that the binding of Con A is specific to the mannoside headgroup. The kinetics of the binding fits to the first order bimolecular reaction model. Fluorescence quenching of fluorescein isothiocyanate labeled Con A after injection into the subphase gives a direct proof of the molecular recognition. (+info)
The crystal structures of Man(alpha1-3)Man(alpha1-O)Me and Man(alpha1-6)Man(alpha1-O)Me in complex with concanavalin A.
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The crystal structures of concanavalin A in complex with Man(alpha1-6)Man(alpha1-O)Me and Man(alpha1-3)Man(alpha1-O)Me were determined at resolutions of 2.0 and 2.8 A, respectively. In both structures, the O-1-linked mannose binds in the conserved monosaccharide-binding site. The O-3-linked mannose of Man(alpha1-3)Man(alpha1-O)Me binds in the hydrophobic subsite formed by Tyr-12, Tyr-100, and Leu-99. The shielding of a hydrophobic surface is consistent with the associated large heat capacity change. The O-6-linked mannose of Man(alpha1-6)Man(alpha1-O)Me binds in the same subsite formed by Tyr-12 and Asp-16 as the reducing mannose of the highly specific trimannose Man(alpha1-3)[Man(alpha1-6)]Man(alpha1-O)Me. However, it is much less tightly bound. Its O-2 hydroxyl makes no hydrogen bond with the conserved water 1. Water 1 is present in all the sugar-containing concanavalin A structures and increases the complementarity between the protein-binding surface and the sugar, but is not necessarily a hydrogen-bonding partner. A water analysis of the carbohydrate-binding site revealed a conserved water molecule replacing O-4 on the alpha1-3-linked arm of the trimannose. No such water is found for the reducing or O-6-linked mannose. Our data indicate that the central mannose of Man(alpha1-3)[Man(alpha1-6)]Man(alpha1-O)Me primarily functions as a hinge between the two outer subsites. (+info)
The pimB gene of Mycobacterium tuberculosis encodes a mannosyltransferase involved in lipoarabinomannan biosynthesis.
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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. (+info)
Biosynthesis of glycosylphosphatidylinositols of Plasmodium falciparum in a cell-free incubation system: inositol acylation is needed for mannosylation of glycosylphosphatidylinositols.
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The structures of glycosylphosphatidylinositols (GPIs) in Plasmodium have been described [Gerold, Schuppert and Schwarz (1994) J. Biol. Chem. 269, 2597-2606]. A detailed understanding of GPI synthesis in Plasmodium is a prerequisite for identifying differences present in biosynthetic pathways of parasites and host cells. A comparison of the biosynthetic pathway of GPIs has revealed differences between mammalian cells and parasitic protozoans. A cell-free incubation system prepared from asexual erythrocytic stages of Plasmodium falciparum, the causative agent of malaria in humans, is capable of synthesizing the same spectrum of GPIs as that found in metabolically labelled parasites. The formation of mannosylated GPIs in the cell-free system is shown to be inhibited by GTP and, unexpectedly, micromolar concentrations of GDP-Man. Lower concentrations of GDP-Man affect the spectrum of GPIs synthesized. The inositol ring of GPIs of P. falciparum is modified by an acyl group. The preferred donor of this fatty acid at the inositol ring is myristoyl-CoA. Inositol acylation has to precede the mannosylation of GPIs because, in the absence of acyl-CoA or CoA, mannosylated GPIs were not detected. Inositol myristoylation is a unique feature of plasmodial GPIs and thus might provide a potential target for drug therapy. (+info)
Candida albicans-derived beta-1,2-linked mannooligosaccharides induce desensitization of macrophages.
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Candida albicans beta-1,2-oligomannosides stimulate macrophage tumor necrosis factor alpha (TNF-alpha) but not NO release. This stimulation desensitized macrophages by altering beta-1, 2-oligomannoside-dependent TNF-alpha production and lipopolysaccharide-dependent TNF-alpha and NO secretion. Desensitization was not related to tyrosine phosphorylation signal transduction but was transferred by culture supernatants in which arachidonic acid derivatives were evidenced. (+info)
The accumulation of Man(6)GlcNAc(2)-PP-dolichol in the Saccharomyces cerevisiae Deltaalg9 mutant reveals a regulatory role for the Alg3p alpha1,3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing.
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N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man. This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step. The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase. (+info)