Structure of the lipid moiety of the Leishmania donovani lipophosphoglycan

The lipid moiety of the lipophosphoglycan of Leishmania donovani had been isolated and characterized as a novel lyso-alkylphosphatidylinositol. Treatment of lipophosphoglycan with either 10% NH4OH or a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus liberated a monoalkylglycerol substituent. Structural characterization of the monoalkylglycerol by gas-liquid chromatography-mass spectrometry indicated the presence of two saturated, unbranched hydrocarbons: a C24 alkyl chain comprising 78% of the lipid with the remaining 22% as a C26 alkyl chain. Periodate sensitivity demonstrated that the alkyl side chain is linked to the C-1 position of the glycerol backbone. Treatment of lipophosphoglycan with nitrous acid released 1-O-alkylglycerophosphorylinositol due to an unacetylated glucosamine residue linked to the inositol of the lyso-alkylphosphatidylinositol. Quantitative analysis of the organic solvent-soluble product of nitrous acid deamination of lipophosphoglycan confirmed the expected ratio of inositol:phosphate:1-O-alkylglycerol as 1:1:1. These results suggest that L. donovani anchors its lipophosphoglycan with a unique lipid component.     

Structure of the lipophosphoglycan from Leishmania major

The major cell surface glycoconjugate of the parasitic protozoan Leishmania major is a heterogeneous lipophosphoglycan. It has a tripartite structure, consisting of a phosphoglycan (Mr 5,000-40,000), a variably phosphorylated hexasaccharide glycan core, and a lysoalkylphosphatidylinositol (lysoalkyl-PI) lipid anchor. The structures of the phosphoglycan and the hexasaccharide core were determined by monosaccharide analysis, methylation analysis, fast atom bombardment-mass spectrometry, one- and two-dimensional 500-MHz (correlated spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA] 1H NMR spectroscopy, and exoglycosidase digestions. The phosphoglycan consists of eight types of phosphorylated oligosaccharide repeats which have the general structure, [formula: see text] where R = H, Galp(beta 1-3), Galp(beta 1-3)Galp(beta 1-3), Arap(alpha 1-2)Galp(beta 1-3), Glcp(beta 1-3)Galp(beta 1-3), Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3), Arap(alpha 1-2)Galp(beta 1-3)Galp(beta 1-3), or Arap(alpha 1-2)Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3), and where all the monosaccharides, including arabinose, are in the D-configuration. The average number of repeat units/molecule (n) is 27. Data are presented which suggest that the nonreducing terminus of the phosphoglycan is capped exclusively with the neutral disaccharide Manp(alpha 1-2)Manp alpha 1-. The structure of the glycan core was determined to be, [formula: see text] where approximately 60% of the mannose residues distal to the glucosamine are phosphorylated and where the inositol is part of the lysoalkyl-PI lipid moiety containing predominantly 24:0 and 26:0 alkyl chains. The unusual galactofuranose residue is in the beta-configuration, correcting a previous report where this residue was identified as alpha Galf. Although most of the phosphorylated repeat units are attached to the terminal galactose 6-phosphate of the core to form a linear lipophosphoglycan (LPG) molecule, some of the mannose 6-phosphate residues may also be substituted to form a Y-shaped molecule. The L. major LPG is more complex than the previously characterized LPG from Leishmania donovani, although both LPGs have the same repeating backbone structure and glycolipid anchor. Finally we show that the LPG anchor is structurally related to the major glycolipid species of L. major, indicating that some of these glycolipids may have a function as precursors to LPG.     

Structure of the phosphosaccharide-inositol core of the Leishmania donovani lipophosphoglycan

The phosphosaccharide-inositol core of the lipophosphoglycan of Leishmania donovani was generated by treatment of the glycoconjugate with mild acid and digestion with phosphatidylinositol-specific phospholipase C. The core was purified and examined by one- and two-dimensional 1H-1H NMR and by methylation analysis. From the results, the carbohydrate core was elucidated as a phosphosaccharide attached to the inositol residue of the lyso-alkylphosphatidylinositol anchor of lipophosphoglycan as follows: PO4----6GalP(alpha 1----6)GalP(alpha 1----3)Galf(alpha 1----3)ManP(alpha 1----3)ManP(alpha 1----4)GlcNP(alpha 1----6)myo-inositol. The presence of an internal galactofuranose residue is highly unusual and the ManP(alpha 1----4)GlcNP(alpha 1----6)myo-inositol sequence is homologous to the respective portion of the glycosylphosphatidylinositol anchors reported for both the Trypanosoma brucei variant surface glycoprotein and the rat brain Thy-1 glycoprotein.     

Refined structure of the lipophosphoglycan of Leishmania donovani.

The primary structure of the major surface glycoconjugate of Leishmania donovani parasites, a lipophosphoglycan, has been further characterized. The repeating PO4-6Galp beta 1-4Man disaccharide units, which are a salient feature of the molecule, are shown to terminate with one of several neutral structures, the most abundant of which is the branched trisaccharide Galp beta 1-4(Manp alpha 1-2)Man. The phosphosaccharide core of lipophosphoglycan, which links the disaccharide repeats to a lipid anchor, contains 2 phosphate residues. One of the core phosphates has previously been localized on O-6 of the galactosyl residue distal to the lipid anchor; the second phosphate is now shown to be on O-6 of the mannosyl residue distal to the anchor and to bear an alpha-linked glucopyranosyl residue. Also, the anomeric configuration of the unusual 3-substituted Galf residue in the phosphosaccharide core is established as beta. The complete structure of the core is thus PO4-6Galp alpha 1-6Galp alpha 1-3Galf beta 1-3[Glcp alpha 1-PO4-6]Manp alpha 1-3Manp alpha 1-4GlcN alpha 1-. This further clarification of the structure of lipophosphoglycan may prove beneficial in determining the structure-function relationships of this highly unusual glycoconjugate.     

Solution structure of the lipophosphoglycan of Leishmania donovani.

The three-dimensional solution structure of the repeating -PO4-6Gal beta 1-4Man alpha 1- disaccharide fragment of the lipophosphoglycan (LPG) derived from Leishmania donovani has been determined by use of a combination of homo- and heteronuclear NMR spin coupling constant measurements together with restrained molecular mechanical minimization and molecular dynamics simulations. The fragment exists with limited mobility in solution about the Gal beta 1-4Man linkages, whereas in contrast a variety of stable rotamers exist about the Man alpha 1-PO4-6Gal linkages. These rotamers define several major stable conformers in solution, which are discussed in terms of the proposed biological role of LPG.     

Structure and antigenicity of the lipophosphoglycan from Leishmania major amastigotes.

The lipophosphoglycan (LPG) of the intracellular amastigote form of the protozoan parasite Leishmania major is chemically distinct from the LPG on the surface of the extracellular promastigote form. Amastigote LPG is composed of the monosaccharides galactose, glucose, mannose, glucosamine and inositol in the molar ratio 51:30:24:1:1; arabinose is absent. The lipid anchor comprises four alkylglycerols, with alkyl chain lengths 24:0, 22:0, 20:0 and 26:0 in the molar ratio 68:18:8:6. Phosphate is present at 4% w/w of total carbohydrate. HPLC gel permeation reveals LPG to be a polydisperse family of molecules Mr 100-6 kDa. The results from immunological studies with LPG-directed antibodies are consistent with amastigote LPG having the expected tripartite structure of GPI-anchor, a core glycan and the phosphorylated disaccharide repeat backbone. Human sera from L. major patients bound amastigote LPG in enzyme-linked immunosorbent assays.     

Functional aspects of the Leishmania donovani lipophosphoglycan during macrophage infection

The most abundant surface glycoconjugate of the Leishmania promastigotes is lipophosphoglycan, a glycosylphosphatidyl-inositol-anchored polymer of the repeating disaccharide-phosphate Gal(beta1,4)Manalpha1-PO4 unit. This complex molecule possesses properties that contribute to the ability of Leishmania to modulate macrophage signaling pathways during the initiation of infection.     

Inhibition of protein kinase C activity by the Leishmania donovani lipophosphoglycan

Purified lipophosphoglycan from Leishmania donovani was found to inhibit the activity of protein kinase C isolated from rat brain. Protein kinase C inhibition by lipophosphoglycan was continuous for 30 minutes. The glycoconjugate was a competitive inhibitor with respect to diolein, a noncompetitive inhibitor with respect to phosphatidylserine, and had no significant effect on protein kinase M and protein kinase A. A possible physiological role of lipophosphoglycan as a negative effector of protein kinase C is suggested.     

Leishmania donovani lipophosphoglycan blocks NADPH oxidase assembly at the phagosome membrane

Phagocytosis of Leishmania donovani promastigotes is characterized by an inhibition of phagolysosome biogenesis mediated by the surface glycolipid lipophosphoglycan (LPG). However, the consequences of this inhibition on macrophage function remain to be determined. In this study, we investigated the impact of LPG-mediated phagosome remodelling on the assembly and function of the NADPH oxidase complex. Phagocytosis of both wild-type and LPG-defective L. donovani promastigotes triggered the release of similar levels of superoxide. However, wild-type promastigotes, but not LPG-defective mutants, inhibited generation of superoxide at the phagosome. Confocal microscopy imaging revealed that the membrane component gp91(phox) and the Rho-family GTPase Rac1 were present on phagosomes containing either wild-type or LPG-defective promastigotes. In contrast, the NADPH oxidase cytosolic components p47(phox) and p67(phox) were excluded from phagosomes in a LPG-dependent fashion. This inhibition is not the consequence of a general defect in the initiation of the NADPH oxidase activation process because both wild-type and LPG-defective promastigotes induced p47(phox) phosphorylation and the formation of complexes containing p47(phox) and p67(phox). Thus, by remodelling their intracellular habitat, L. donovani promastigotes prevent the assembly of a functional phagosomal NADPH oxidase complex, thereby evading an important host innate defence mechanism.     

Leishmania donovani lipophosphoglycan disrupts phagosome microdomains in J774 macrophages

Clearance of pathogens by phagocytosis and their killing in phagolysosomes is a key aspect of our innate ability to fight infectious agents. Leishmania parasites have evolved ways to survive and replicate in macrophages by inhibiting phagosome maturation and avoiding the harsh environment of phagolysosomes. We describe here that during this process Leishmania donovani uses a novel strategy involving its surface lipophosphoglycan (LPG), a virulence factor impeding many host functions, to prevent the formation or disrupt lipid microdomains on the phagosome membrane. LPG acts locally on the membrane and requires its repetitive carbohydrate moieties to alter the organization of microdomains. Targeting and disruption of functional foci, where proteins involved in key aspects of phagolysosome biogenesis assemble, is likely to confer a survival advantage to the parasite.     

Leishmania donovani lipophosphoglycan selectively inhibits signal transduction in macrophages.

The effect of purified lipophosphoglycan (LPG) of Leishmania donovani on signal transduction and gene expression in murine bone marrow-derived macrophages was investigated. LPG stimulated the rapid expression of both c-fos and TNF genes within 30 min after exposure, suggesting that the interaction of LPG with its receptor stimulated a specific signal transduction pathway. Macrophages pretreated with LPG for 3 h became unresponsive to subsequent stimulation with LPS and the activators of protein kinase C, 1-oleoyl-2-acetyl-glycerol, and calcium ionophore A23187. Moreover, LPG induced a rapid down-modulation of TNF receptors. In contrast, the ability of macrophages to express the c-fos gene in response to the cAMP analogue, dibutyryl cAMP, was not impaired by LPG. Fragmentation of LPG revealed that the inhibitory activity of LPG required both the repeating phosphorylated disaccharides and the phosphosaccharide core. Collectively, these data demonstrate that LPG selectively impaired signal transduction in macrophages and suggest a role for this molecule in the survival of the parasite within the macrophage.     

Structure of Leishmania mexicana lipophosphoglycan.

Lipophosphoglycan (LPG) was isolated from the culture supernatant of Leishmania mexicana promastigotes and its structure elucidated by a combination of 1H NMR, fast atom bombardment mass spectrometry, methylation analysis, and chemical and enzymatic modifications. It consists of the repeating phosphorylated oligosaccharides PO4-6Gal beta 1-4Man alpha 1- and PO4-6[Glc beta 1-3]Gal beta 1-4Man alpha 1-, which are linked together in linear chains by phosphodiester linkages. Each chain of repeat units is linked to a phosphosaccharide core with the structure PO4-6Gal alpha 1-6Gal alpha 1-3Galf beta 1- 3[Glc alpha 1-PO4-6]Man alpha 1-3Man alpha 1-4GlcNH2 alpha 1-6 myo-inositol, where the myo-inositol residue forms the head group of a lyso-alkylphosphatidylinositol moiety. The nonreducing terminus of the repeat chains appear to be capped with the neutral oligosaccharides Man alpha 1-2Man, Man alpha 1-2Man alpha 1-2Man, or Man alpha 1-2[Gal beta 1-4]Man. Cellular LPG, isolated from promastigotes, has a very similar structure to the culture supernatant LPG. However, it differs from culture supernatant LPG in the average number of phosphorylated oligosaccharide repeat units (20 versus 28) and in alkyl chain composition. Although culture supernatant LPG contained predominantly C24:0 alkyl chains, cellular LPG contained approximately equal amounts of C24:0 and C26:0 alkyl chains. It is suggested that culture supernatant LPG is passively shed from promastigotes and that it may contribute significantly, but not exclusively, to the "excreted factor" used for serotyping Leishmania spp. Comparison of L. mexicana LPG with the LPGs of Leishmania major and Leishmania donovani indicate that these molecules are highly conserved but that species-specific differences occur in the phosphorylated oligosaccharide repeat branches and in the relative abundance of the neutral cap structures.     

Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts

Lipophosphoglycan (LPG), the major surface glycoconjugate on Leishmania donovani promastigotes, is crucial for the establishment of infection inside macrophages. LPG comprises a polymer of repeating Galβ1,4Manα-PO₄ attached to a lysophosphatidylinositol membrane anchor. LPG is transferred from the parasite to the host macrophage membrane during phagocytosis and induces periphagosomal F-actin accumulation correlating with an inhibition of phagosomal maturation. The biophysical properties of LPG suggest that it may be intercalated into membrane rafts of the host-cell membrane. The aim of this study was to investigate if the effects of LPG on phagosomal maturation are mediated via action on membrane rafts. We show that LPG accumulates in rafts during phagocytosis of L. donovani and that disruption of membrane rafts abolished the effects of LPG on periphagosomal F-actin and phagosomal maturation, indicating that LPG requires intact membrane rafts to manipulate host-cell functions. We conclude that LPG associates with membrane rafts in the host cell and exert its actions on host-cell actin and phagosomal maturation through subversion of raft function.     

Requirement of lipophosphoglycan for intracellular survival of Leishmania donovani within human monocytes

The function of the lipophosphoglycan of Leishmania donovani parasites was investigated in human peripheral monocytes. In contrast to wild-type L. donovani which grow in monocytes, incubation of monocytes with two variant lines of L. donovani, defective in lipophosphoglycan expression, resulted in the entry of the variant cells into the monocytes and their subsequent destruction. Passive transfer of lipophosphoglycan to the variant cells led to prolonged survival in monocytes. These results indicate that lipophosphoglycan is required by the parasite for intracellular survival. To investigate one possible protective role of the glycoconjugate, preincubation of monocytes with a suspension of lipophosphoglycan and subsequent treatment of the cells with PMA or opsonized zymosan resulted in an attenuation of the oxidative burst; the attenuation effect was concentration dependent on the glycoconjugate and independent of preincubation time. Moreover, hydrophobic beads, coated with lipophosphoglycan, were phagocytized by monocytes and found to inhibit oxygen consumption in monocytes activated with PMA. These results suggest a possible relationship between the absence of lipophosphoglycan in the variant parasites and their inability to survive within monocytes. Although the precise molecular basis remains to be elucidated, the ability of lipophosphoglycan to impair the microbial oxidative response may be a contributing factor in its requirement for intracellular survival.     

Effect of Leishmania donovani lipophosphoglycan on ornithine decarboxylase activity in macrophages.

Lipophosphoglycan (LPG), a major surface molecule from Leishmania donovani, stimulated ornithine decarboxylase (ODC) activity in macrophages in a dose- and time-dependent manner. LPG stimulated the rapid increase in ODC activity within 30 min after exposure, suggesting that the interaction of LPG with its receptor stimulated a specific signal transduction pathway. However, LPG-induced ODC activity was a transient event because 3 hr after exposure to LPG, no stimulation of ODC activity was detectable. ODC activity appeared to be coupled to the activation of protein kinase C (PKC) in macrophages, as activators of PKC caused a rapid increase in the ODC activity. Macrophages pretreated with LPG for 1 hr became unresponsive to subsequent stimulation by the PKC activators 1-oleoyl-2-acetyl-glycerol and the calcium ionophore A23187. In contrast, the ability of macrophages to express ODC activity in response to the cyclic AMP analogue dibutyryl cyclic AMP was not impaired by LPG.     

Enzymes of carbohydrate metabolism in Leishmania donovani amastigotes

A method for the isolation of Leishmania donovani amastigotes from infected hamster spleen and liver tissues is described. Over 85% of the isolated amastigotes were viable as judged by acridine orange-ethidium bromide staining and in vitro transformation to the promastigote form. A comprehensive survey of the enzymes of carbohydrate metabolism in L. donovani amastigotes and promastigotes was conducted. Amastigotes and promastigotes possess all of the enzymes of the Embden-Meyerhof pathway, hexose monophosphate shunt, and tricarboxylic acid cycle. Cell-free extracts of both forms demonstrate an active glutamate dehydrogenase, thus linking activity which permits entry of pyruvate into the tricarboxylic acid cycle. Both forms demonstrate an active glutamate dehydrogenase, thus linking amino acid metabolism with carbohydrate metabolism. Pyruvate carboxylase, the enzyme responsible for replenishment of C4 acids by heterotrophic CO2 fixation into pyruvate, was also demonstrable in the tissue and insect forms. In general, activities of promastigote enzymes are higher than the amastigote enzymes. Differences between the vertebrate (amastigote) and invertebrate (promastigote) forms in their potential to utilize carbohydrates as substrates would appear to be quantitative rather than qualitative.     

AuBr3 mediated glycosidations: synthesis of tetrasaccharide motif of the Leishmania donovani lipophosphoglycan

Tetrasaccharide cap present in lipophosphoglycan of the Leishmania donovani responsible for visceral Leishmaniaisis is synthesized as a fully protected propargyl glycoside. AuBr(3) mediated selective glycosylation of propargyl 1,2-orthoester in the presence of propargyl glycoside is employed as a key step to obtain propargyl containing oligomers. Further, propargyl tetrasaccharide is connected with a long chain hydrocarbon containing azidothiol functionality situated at two terminal ends via 'click' reaction.     

Characterization of the glucosyltransferases that assemble the side chains of the Indian Leishmania donovani lipophosphoglycan

The bacterium Bacillus sp. GL1 assimilates two kinds of heteropolysaccharides, gellan and xanthan, by using extracellular gellan and xanthan lyases, respectively, and produces unsaturated saccharides as the first degradation products. A novel unsaturated glucuronyl hydrolase (glycuronidase), which was induced in the bacterial cells grown on either gellan or xanthan, was found to act on the tetrasaccharide of unsaturated glucuronyl-glucosyl-rhamnosyl-glucose produced from gellan by gellan lyase, and the enzyme and its gene were isolated from gellan-grown cells. The nucleotide sequence showed that the gene contained an ORF consisting of 1131 base pairs coding a polypeptide with a molecular weight of 42,859. The purified enzyme was a monomer with a molecular mass of 42 kDa and was most active at pH 6.0 and 45 degrees C. Because the enzyme can act not only on the gellan-degrading product by gellan lyase, but also on unsaturated chondroitin and hyaluronate disaccharides produced by chondroitin and hyaluronate lyases, respectively, it is considered that the unsaturated glucuronyl hydrolase plays specific and ubiquitous roles in the degradation of oligosaccharides with unsaturated uronic acid at the nonreducing terminal produced by polysaccharide lyases.     

Characterization of lipophosphoglycan from a Ricin-resistant mutant of Leishmania major

One of the virulence factors of the protozoan parasite Leishmaniamajor is the surface glycoconjugate, lipophosphoglycan (LPG).A Ricin-resistant mutant of L.major was generated and characterisedwith respect to its virulence in mice and the structure andexpression of LPG. The LPG from this mutant (1F6-B5) retainedthe tripartite structure of wild-type LPG, comprising a glycosylphosphatidylinositol(GPI) anchor linked to a phosphorylated disaccharide backboneterminating in a nonreducing neutral oligosaccharide cap. Thestructure of the GPI anchor and the major capping oligosaccharidewere identical to wild-type LPG. However, there were variationsin the number of phosphorylated repeats (PO₄-6Gal(ß1-4)Man(     

The Leishmania donovani lipophosphoglycan T lymphocyte-reactive component is a tightly associated protein complex

Lymphocytes from mice immunized with Leishmania donovani (LPG) were specifically stimulated to proliferate in vitro by purified LPG or its delipidated congener, phosphoglycan. The response was dose dependent and required prior immunization with either LPG or phosphoglycan. Proliferation was eliminated by specific depletion of Thy-1+ cells with antisera and C and the proliferating T cell subset was shown to be CD4+CD8-. Tests of various LPG fragments indicated that the T cell stimulation was associated with the core structure of LPG rather than the lipid or phosphoglycan repeat structure. However, amino acid analysis of LPG and active LPG fragments, after acid hydrolysis, showed the presence of amino acids in peptide linkage. Specific hydrolysis of the glycosidic linkages in LPG with trifluoromethanesulfonic acid provided polypeptide material reactive with two mAb previously believed to be LPG carbohydrate core specific. The protein was separated from LPG by reverse phase chromatography and shown to be a complex of proteins with common epitopes recognized by the two mAb. The dominant species isolated from LPG was a set of small, approximately 11,000 Mr, molecules. Subsequent T cell proliferation studies showed that the lymphocyte stimulation was associated with the protein component of LPG and not the glycan.