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To study the function of side chain oligosaccharides of the cell-surface lipophosphoglycan (LPG), mutagenized Leishmania major defective in side chain biosynthesis were negatively selected by agglutination with the monoclonal antibody WIC79.3, which recognizes the galactose-containing side chains of L. major LPG. One such mutant, called Spock, lacked the ability to bind significantly to midguts of the natural L. major vector, Phlebotomus papatasi, and to maintain infection in the sand fly after excretion of the digested bloodmeal. Biochemical characterization of Spock LPG revealed its structural similarity to the LPG of Leishmania donovani, a species whose inability to bind to and maintain infections in P. papatasi midguts has been strongly correlated with the expression of a surface LPG lacking galactose-terminated oligosaccharide side chains. An in vitro galactosyltransferase assay using wild-type or Spock membranes was used to determine that the defect in Spock LPG biosynthesis is a result of defective beta1,3-galactosyltransferase activity as opposed to a modification of LPG, which would prevent it from serving as a competent substrate for galactose addition. The results of these experiments show that Spock lacks the beta1, 3-galactosyltransferase for side chain addition and that the LPG side chains are required for L. major to bind to and to produce transmissible infection in P. papatasi.

We have previously established that lipopolysaccharide (LPS) induces the expression of new specific LPS-binding sites (LpsR) in mouse bone marrow cells (BMC). We now show that exposure of human BMC to LPS elicits the production of both CD14 molecules (detectable with monoclonal antibody My4) and LpsR (detectable with fluorescein isothiocyanate-LPS). Pretreatment of stimulated human BMC with My4 inhibited the binding of fluorescein isothiocyanate-LPS. The stimulation of human BMC, but not mouse BMC, required the presence of serum. Other characteristics of mouse and human BMC examined were very similar. Their inducible LpsR interacted with the lipid moieties of LPS and Leishmania donovani lipophosphoglycan and with a soluble preparation of peptidoglycan. Moreover, mouse and human LpsR were susceptible to treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), thus suggesting that both are PI-anchored CD14 molecules. Neither LpsR appeared able to interact with a synthetic LPS antagonist (compound PPDm2) structurally related to the lipid region of LPS. However, PPDm2 blocked LPS-induced expression of LpsR in both BMC. Furthermore, in both species, pretreatment of BMC with PI-PLC did not prevent the cells from expressing LpsR in response to LPS. The results support the hypothesis that the elicited LpsR of mouse and human BMC is an inducible form of CD14, whereas the putative "signaling LPS receptor" of these cells is not CD14 or any other PI-anchored molecule.

A mutant cell line of Leishmania donovani (R2D2), previously selected for resistance to the cytotoxic lectin ricin agglutinin, was found to be totally deficient in the synthesis and expression of lipophosphoglycan, a dominant surface virulence factor. The metabolic defect in R2D2 parasites responsible for its lipophosphoglycan (LPG-) phenotype was investigated in this study. Following metabolic labeling of R2D2 parasites with either [3H]galactose or [3H]mannose, the main glycosylphosphatidylinositide product that accumulated was Glc-PO4-Man-Man-GlcN-lyso-1-O-alkylphosphatidylinositol (PI). The metabolic defect was further defined using a cell-free glycosylation system. When membrane preparations from wild-type cells were incubated with UDP-[3H]galactose and unlabeled GDP-mannose in the absence of exogenous acceptors, radiolabeled lipophosphoglycan was synthesized. The addition of exogenous Man-Man-GlcN-PI or Galf-Man-Man-GN-PI stimulated lipophosphoglycan synthesis in vitro. In contrast, when membrane preparations from R2D2 cells were incubated with exogenous Man-Man-GlcN-PI as an acceptor or in the absence of exogenous acceptor, the truncated glycosylphosphatidylinositide Glc-PO4-Man-Man-GlcN-PI was the main radioactive product synthesized. However, when exogenous Galf-Man-Man-GN-PI was added to the R2D2 in vitro system, radioactive lipophosphoglycan was synthesized. Collectively, these results indicate that the mutant R2D2 cells are unable to complete the assembly of the glycan core of LPG because of a defect in the synthesis of the "activated" galactofuranosyl donor or the lack of a functional galactofuranosyltransferase.

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.

An extracellular phosphoglycan (exPG), present in the culture medium of the promastigote form of Leishmania donovani, was purified and structurally characterized. The purification scheme included ethanol precipitation of the culture medium, anion exchange chromatography, hydrophobic chromatography on phenyl-Sepharose, and preparative polyacrylamide gel electrophoresis. Structural analysis by 1H-1H NMR, methylation linkage analysis, and glycosidase digestion revealed that the exPG consisted of the following structure: (CAP)----[PO4-6Galp beta 1-4Manp alpha 1]10-11-PO4-6Galp beta 1-4Man. The cap was found to be one of several small, neutral oligosaccharides, the most abundant of which was the trisaccharide Galp beta 1-4(Manp alpha 1-2)Man. The results indicated structural analogy to the cellular-derived lipophosphoglycan (LPG) from L. donovani. The important exceptions are a lack of the lipid anchor, the entire phosphosaccharide core, and several of the repeating disaccharide units. Although the function of exPG is presently unknown, it may play a protective role for the promastigote in the insect vector or during infection of a mammalian host.

Incubation of microsomal preparations from Leishmania donovani parasites with UDP-[3H]galactose or GDP-[14C]mannose resulted in incorporation of radiolabel into an endogenous product that exhibited the chemical and chromatographic characteristics of the parasite's major surface glycoconjugate, lipophosphoglycan. The [3H]galactose- or [14C]mannose-labeled product was (i) cleaved by phosphatidylinositol-specific phospholipase C; (ii) deaminated by nitrous acid; and (iii) degraded into radioactive, low molecular weight fragments upon hydrolysis with mild acid. Analysis of the products of mild acid hydrolysis revealed the presence of phosphorylated Gal-beta-Man as the major fragment with lesser amounts of mono-, tri-, and tetrasaccharides. The incorporation of the two isotopic precursors was neither stimulated by the addition of dolichylphosphate nor inhibited by amphomycin, indicating that dolichol-saccharide intermediates are not involved in assembly of the repeating units of lipophosphoglycan. Development of this cell-free glycosylating system will facilitate further studies on the pathway and enzymes involved in lipophosphoglycan biosynthesis.

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.

Total glycopeptides from human K-562 cells, labeled metabolically with [3H]glucosamine or [3H]mannose, were prepared by extracting the cells with organic solvents to remove lipids and by digesting the residue with pronase. 3H-labeled glycopeptides were fractionated on Sephadex G-50 revealing a high molecular weight fraction (Mr = 7,000 to 11,000), comprising approximately 10% of the [3H]glucosamine and 25% of the [3H]mannose label. Digestion of this glycopeptide fraction with endo-beta-galactosidase from Escherichia freundii, specific for a repeating structure of Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 3), results in the following four products as resolved by Bio-Gel P-2 gel filtration: 1) a disaccharide with the structure beta-2-deoxy-2-acetamidoglucosyl leads to beta-galactose; 2) a trisaccharide with the structure beta-galactosyl leads to beta-2-deoxy-2-acetamidoglucosyl leads to beta-galactose; 3) a tetrasaccharide with the sequence alpha-N-acetylneuraminyl leads to beta-galactosyl leads to beta-2-deoxy-2-acetamidoglucosyl leads to beta-galactose; and 4) a larger, complex fragment which contains mannose and beta-2-deoxy-2-acetamidoglucose and which is probably the protein linkage region. In addition, visualization of radiolabeled glycoproteins by fluorography on polyacrylamide gels revealed a 105,000-dalton "Band 3"-like glycoprotein and other bands that were sensitive to endo-beta-galactosidase. These results indicate that the K-562 cell line bears a glycopeptide, erythroglycan, which has been found on erythrocytes, and that this polymer is expressed mainly in the fetal form as a linear chain.

Following the rapid enzymatic transfer of an oligosaccharide (GlcNAc2Man9Glc3) from a lipid carrier to endogenous protein acceptors in membrane preparations from NIL fibroblasts, the transferred oligosaccharide chain undergoes processing. Protein-bound oligosaccharides, released from the polypeptide backbone by treatment with endo-beta-N-acetylglucosaminidase H, were analyzed by gel filtration and by susceptibility to alpha-mannosidase digestion. The initial stages of this processing in vitro consist of sequential excision of 3 glucose residues prior to the removal of mannose residues. The array of oligosaccharides generated in vitro by membrane preparations from NIL cells appears to be identical with processed oligosaccharides derived in vivo in intact NIL cells.

The lipid-linked oligosaccharide synthesized in vitro, in the presence of 1.0 microM UDP-[3H]Glc, GDP-[14C]Man, and UDP-GlcNAc has been isolated and the structure of the oligosaccharide has been analyzed. The oligosaccharide contains 2 N-acetylglucosamine, 9 mannose, and 3 glucose residues. The N-acetylglucosamine residues are located at the reducing terminus. The 3 glucose residues are arranged in a linear order at one of the nonreducing termini in the sequence Glc 1,2--Glc 1,3--Glc--(Man)9 (GlcNAc)2. The structural analysis was made possible largely by the availability of glucosidase preparations of fungal anad microsomal origin which remove glucose residues from the oligosaccharide without releasing mannose residues.