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.     

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.     

A family of glycoinositol phospholipids from Leishmania major. Isolation, characterization, and antigenicity

The glycolipids of the protozoan Leishmania major strain LRC-L119 belong to a class of glycoinositol phospholipids (GIPL) that show partial structural homology to the phosphatidylinositol-containing glycolipid membrane anchors of several eukaryotic proteins and the lipid moiety of L. major lipophosphoglycan. The GIPLs were the only glycolipids detected and were purified by octyl-Sepharose and thin layer chromatographies. Analysis of the native and dephosphorylated glycolipids (GIPLs 1-6) by gas chromatography-mass spectrometry revealed that the glycan moieties have between 4 and 10 saccharide residues and all contain mannose, galactose, and non-N-acetylated glucosamine. Some of the GIPLs also contain glucose (GIPL-6) and hexose monophosphate residues (GIPL 4-6). The presence of an inositol phospholipid moiety in all the GIPLs is indicated by the identification of 1 myo-inositol monophosphate residue/molecule and their susceptibility to phosphatidylinositol-specific phospholipase C. However, heterogeneity in the lipid moieties is indicated by differences in the compositional analysis and the behavior of the GIPLs on the thin layer chromatography after mild alkali hydrolysis or phospholipase A2 treatment. These results demonstrate that GIPLs 1-4 contain 1-alkyl-2-acylglycerol composed of saturated unbranched alkyl chains with carbon chain lengths of 18-26 and acyl chains of myristate, palmitate and stearate, whereas GIPL-5 and -6 contain lyso-alkylglycerol composed of mainly C24:0 and C26:0 alkyl chains. Analysis of the products of nitrous acid deamination demonstrates that these glycerolipids are present as alkylacylphosphatidylinositol (GIPLs 1-4) and 1-O-alkylglycerophosphoinositol (GIPL-5 and -6), respectively. GIPL-2 and -3 are labeled on the surface of living promastigotes with galactose oxidase/NaB[3H]4. These GIPLs also react with three monoclonal antibodies that recognize the surface of promastigotes and amastigotes of L. major and other Leishmania spp.     

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus

Acidic glycosphingolipid components were extracted from the opportunistic mycopathogen Aspergillus fumigatus and identified as inositol phosphorylceramide and glycosylinositol phosphorylceramides (GIPCs). Using nuclear magnetic resonance sppectroscopy, mass spectrometry, and other techniques, the structures of six major components were elucidated as Ins-P-Cer (Af-0), Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-2), Manp(alpha1-->2)Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-3a), Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)-Ins-P-Cer (Af-3b), Manp(alpha1-->2)-Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)Ins-P-Cer (Af-4), and Manp(alpha1-->3)Manp(alpha1-->6)GlcpN(alpha1-->2)Ins-P-Cer (Af-3c) (where Ins = myo-inositol and P = phosphodiester). A minor A. fumigatus GIPC was also identified as the N-acetylated version of Af-3c (Af-3c*), which suggests that formation of the GlcNalpha1-->2Ins linkage may proceed by a two-step process, similar to the GlcNalpha1-->6Ins linkage in glycosylphosphatidylinositol (GPI) anchors (transfer of GlcNAc, followed by enzymatic de-N-acetylation). The glycosylinositol of Af-3b, which bears a distinctive branching Galf(beta1-->6) residue, is identical to that of a GIPC isolated previously from the dimorphic mycopathogen Paracoccidioides brasiliensis (designated Pb-3), but components Af-3a and Af-4 have novel structures. Overlay immunostaining of A. fumigatus GIPCs separated on thin-layer chromatograms was used to assess their reactivity against sera from a patient with aspergillosis and against a murine monoclonal antibody (MEST-1) shown previously to react with the Galf(beta1-->6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.     

Developmental changes in the glycosylated phosphatidylinositols of Leishmania donovani. Characterization of the promastigote and amastigote glycolipids.

In addition to utilizing glycosylated phosphatidylinositols (GPIs) as anchors for surface proteins, protozoan parasites of the genus Leishmania synthesize two novel classes of GPI: the polydisperse lipophosphoglycans (LPGs) and a family of low molecular weight glycoinositol phospholipids (GIPLs). We now show that LPG is expressed in high copy number (6 x 10(6) molecules/cell) in the promastigote (insect) stage of L. donovani but not in the amastigote stage, which infects mammalian macrophages. Detection of these molecules was by gas chromatography-mass spectrometric analyses and by a sensitive radiolabeling procedure. In contrast, a novel family of GIPLs was present in high copy number (approximately 10(7) molecules/cell) in both promastigote and amastigote stages of L. donovani. These glycolipids were purified and analyzed by gas chromatography-mass spectrometry, methylation analysis, and by chemical and enzymatic sequencing after deamination and NaB3H4 reduction. Promastigotes contained three major GIPLs species with the following generalized structure [formula: see text] where R = H for isoM2, Man alpha 1- for isoM3 or Man alpha 1-2Man alpha 1- for isoM4. Amastigotes contained two major GIPL species that lacked the alpha 1-3-linked mannose branch and had the linear structures Man alpha 1-6Man alpha 1-4GlcN (M2) and Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN (M3) linked to alkylacyl-PI. The 1-O-alkyl-2-acyl-PI moieties of all these species contained predominantly C18:0 alkyl chains and C16:0 or C18:0 fatty acids. Amastigotes contained, in addition, a GalNAc beta 1-3 terminating glycosphingolipid with homology to the mammalian para Forssman glycolipid. This glycolipid appeared to be a constituent of the parasite membrane but was not metabolically labeled with [3H]glucose, suggesting that it was acquired from host cells. These results suggest that LPG may not be required for amastigote survival in the mammalian host and that the GIPLs are likely to be major components on the surface membrane in both stages.     

Molecular analysis of a novel family of complex glycoinositolphosphoryl ceramides from Cryptococcus neoformans: structural differences between encapsulated and acapsular yeast forms

Complex glycoinositolphosphoryl ceramides (GIPCs) have been purified from a pathogenic encapsulated wild-type (WT) strain of Cryptococcus neoformans var. neoformans and from an acapsular mutant (Cap67). The structures of the GIPCs were determined by a combination of tandem mass spectrometry, nuclear magnetic resonance spectroscopy, methylation analysis, gas chromatography-mass spectrometry, and chemical degradation. The main GIPC from the WT strain had the structure Manp(alpha1-3)[Xylp(beta1-2)] Manp(alpha1-4)Galp(beta1-6)Manp(alpha1-2)Ins-1-phosphoryl ceramide (GIPC A), whereas the compounds from the acapsular mutant were more heterogeneous in their glycan chains, and variants with Manp(alpha1-6) (GIPC B), Manp(alpha1-6) Manp(alpha1-6) (GIPC C), and Manp(alpha1-2)Manp(alpha1-6)Manp(alpha1-6) (GIPC D) substituents linked to the nonreducing terminal mannose residue found in the WT GIPC A were abundant. The ceramide moieties of C. neoformans GIPCs were composed of a C(18) phytosphingosine long-chain base mainly N-acylated with 2-hydroxy-tetracosanoic acid in the WT GIPC while in the acapsular Cap67 mutant GIPCs, as well as 2-hydroxy-tetracosanoic acid, the unusual 2,3-dihydroxy-tetracosanoic acid was characterized. In addition, structural analysis revealed that the amount of GIPC in the WT cells was fourfold less of that in the acapsular mutant.     

Antigenic determinants of bacteria. The structure of the repeating unit of a specific polysaccharide from Shigella boydii type 7

Acid hydrolysis of the antigenic lipopolysaccharide from Shigella boydii type 7 afforded a specific polysaccharide composed of 2-acetamido-2-deoxy-D-glucose, D-glucose, D-galactose, 5-acetamido-3,5,7,9-tetradeoxy-7-[(3R)-3-hydroxybutyramido]-L- glycero-L-manno-nonulosonic acid (NonN2A) and acetic acid residues in the 1:1:2:1:1 ratio. From the results of methylation analysis, hydrogen fluoride solvolysis and Smith degradation, the structure of the repeating unit of the specific polysaccharide was dedused as: -2) Galf (beta 1-3)GlcNAcp (alpha 1-8)NonN2A (beta 2-6) Galp (alpha 1-6) Glcp (alpha 1-4 increases Ac. The 13C NMR spectrum of the polysaccharide was interpreted, and the spectral data fully confirmed the structure of the polysaccharide repeating unit.     

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.     

Structural study of a cell wall mannan-protein complex of the pathogenic yeast Candida glabrata IFO 0622 strain.

We conducted a structural analysis of the cell wall mannan-protein complex (mannan) isolated from a pathogenic yeast, Candida glabrata IFO 0622 strain. The chemical structure of mannobiose released from this mannan by treatment with 10 mM HCl at 100 degrees C for 1 h was identified as Manp beta 1-2Man. The treatment of this mannan with 100 mM NaOH at 25 degrees C for 18 h gave a mixture of alpha-1,2- and alpha-1,3-linked oligosaccharides, from tetraose to biose, and mannose. The acid- and alkali-stable mannan moiety was subjected to mild acetolysis with a 100:100:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4 at 40 degrees C for 36 h. The resultant three novel oligosaccharides, tetraose, hexaose, and heptaose, were identified as Manp beta 1-2Manp alpha 1-2Manp alpha 1-2Man, Manp alpha 1-2Manp alpha 1-2Manp alpha 1-6Manp alpha 1-2Manp alpha 1-2Man, and Manp alpha 1-3Manp alpha 1-2Manp alpha 1-2Manp alpha 1-6Manp alpha 1- 2Manp alpha 1-2Man, respectively, in addition to the three known oligosaccharides, Manp alpha 1-2Man, Manp alpha 1-2Manp alpha 1-2Man, and Manp alpha 1-3Manp alpha 1-2Manp alpha 1-2Man. A sequential analytical procedure involving partial acid hydrolysis with hot 0.3 M H2SO4, methylation, fast atom bombardment mass, and 1H NMR analyses was quite effective in the structural determination of the novel oligosaccharides. The results indicate that this mannan possesses a structure closely resembling that of Saccharomyces cerevisiae X2180-1A wild type strain, with the presence of small amounts of oligomannosyl residue, Manp beta 1-2Manp alpha 1-X, corresponding to one of the epitopes dominating serotype-A specificity of Candida spp., in addition to branches corresponding to hexaose and heptaose each containing one intermediary alpha-1,6 linkage.     

Structural characterization of neutral and acidic glycolipids from Thermus thermophilus HB8

The structural characterization of glycolipids from Thermus thermophilus HB8 was performed in this study. Two neutral and one acidic glycolipids were extracted and purified by the modified TLC-blotting method, after which their chemical structures were determined by chemical composition analysis, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The structure of one of the neutral glycolipids, NGL-A, was Galp(α1-6)GlcpNacyl(β1-2)Glcp(α1-)acyl(2)Gro, and the other, NGL-C, was Galf(β1-2)Galp(α1-6)GlcpNacyl(β1-2)Glcp(α1-)acyl(2)Gro. The structure of NGL-C was identical to that reported previously [Oshima, M. and Ariga, T. (1976) FEBS Lett. 64, 440]. Both neutral glycolipids shared a common structural unit found in the Thermus species. The acyl groups found in NGL-A and NGL-C, iso-type pentadecanoxy and heptadecanoxy fatty acid, were also the same as those found in this species. In contrast, the acidic glycolipid, AGL-B, possessed the structure of N-(((GlcpNAc(α1-)acyl(2)Gro)P-2)GroA)alkylamine. The alkyl group in AGL-B was an iso-type heptadecanyl, suggesting that the iso-type structure of the long alkyl chain is responsible for the thermal stability of the bacteria.     

Polymerization of mycobacterial arabinogalactan and ligation to peptidoglycan

The cell wall of Mycobacterium spp. consists predominately of arabinogalactan chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-(alpha1-3)-Rha linkage unit (LU) and esterified to a variety of mycolic acids at the nonreducing ends. Several aspects of the biosynthesis of this complex have been defined, including the initial formation of the LU on a polyprenyl phosphate (Pol-P) molecule followed by the sequential addition of galactofuranosyl (Galf) units to generate Pol-P-P-LU-(Galf)1,2,3, etc. and Pol-P-P-LU-galactan, catalyzed by a bifunctional galactosyltransferase (Rv3808c) capable of adding alternating 5- and 6-linked Galf units. By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we now demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, finally, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LU-arabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs.     

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.     

Structural study of phosphomannan of yeast-form cells of Candida albicans J-1012 strain with special reference to application of mild acetolysis

Structural analysis of the phosphomannan isolated from yeast-form cells of a pathogenic yeast, Candida albicans J-1012 strain, was conducted. Treatment of this phosphomannan (Fr. J) with 10 mM HCl at 100 degrees C for 60 min gave a mixture of beta-1,2-linked manno-oligosaccharides, from tetraose to biose plus mannose, and an acid-stable mannan moiety (Fr. J-a), which was then acetolyzed by means of an acetolysis medium, 100:100:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4, at 40 degrees C for 36 h in order to avoid cleavage of the beta-1,2 linkage. The resultant manno-oligosaccharide mixture was fractionated on a column of Bio-Gel P-2 to yield insufficiently resolved manno-oligosaccharide fractions higher than pentaose and lower manno-oligosaccharides ranging from tetraose to biose plus mannose. The higher manno-oligosaccharide fraction was then digested with the Arthrobacter GJM-1 alpha-mannosidase in order to cleave the enzyme-susceptible alpha-1,2 and alpha-1,3 linkages, leaving manno-oligosaccharides containing the beta-1,2 linkage at their nonreducing terminal sites, Manp beta 1----2Manp alpha 1----2Manp alpha 1----2Manp alpha 1----2Man, Manp beta 1----2Manp beta 1----2Manp alpha 1----2Manp alpha 1---- 2Manp alpha 1----2Man, and Manp beta 1----2Manp beta 1----2Manp beta 1----2Manp alpha 1---- 2Manp alpha 1----2Manp alpha 1----2Man. However, the result of acetolysis of Fr. J-a by means of a 10:10:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4 at 40 degrees C for 13 h was significantly different from that obtained by the mild acetolysis method; i.e., the amount of mannose was apparently larger than that formed by the mild acetolysis method. In summary, a chemical structure for Fr. J as a highly branched mannan containing 14 different branching moieties was proposed.     

Identification of the defect in lipophosphoglycan biosynthesis in a non-pathogenic strain of Leishmania major.

The major macromolecule on the surface of the protozoan parasite, Leishmania major, is a complex lipophosphoglycan (LPG), which is anchored to the plasma membrane by an inositol-containing phospholipid. A defect in LPG biosynthesis is thought to be responsible for the avirulence of the L. major strain LRC L119 in mice. In order to identify the nature of this defect we have characterized two truncated forms of LPG, which are accumulated in this strain, by one- and two-dimensional 500-MHz 1H NMR spectroscopy, two-dimensional heteronuclear 1H-31P NMR spectroscopy, methylation analysis, and exoglycosidase digestions. The structures of these glycoinositolphospholipids, termed GIPL-4 and -6, are as follows: [formula: see text] The glycan moieties of GIPL-4 and -6 are identical to the anchor region of LPG, which is also substituted with a Glc-1-PO4 residue in approximately 60% of the structures. However, instead of being capped with chains of phosphorylated oligosaccharide repeat units, both glycan moieties terminate in Man alpha 1-PO4, suggesting that the defect in LPG biosynthesis is in the transfer of galactose to this residue to form the disaccharide backbone of the first repeat unit. These results indicate that the phosphoglycan moiety of LPG is essential for intracellular survival of the parasite and have implications for LPG biosynthesis.     

Accumulation of a globo-series glycolipid having Gal alpha 1-3Gal in PC12h pheochromocytoma cells

In a previous paper, we reported the presence of globoside as a major neutral glycolipid in PC12 pheochromocytoma cells [Ariga, T., Macala, L. J., Saito, M., Margolis, R. K., Greene, L. A., Margolis, R. U., & Yu, R. K. (1988) Biochemistry 27, 52-58]. Recently, we found that subcloned PC12h cells accumulated another unusual neutral glycolipid. In order to characterize this glycolipid, PC12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated two major neutral glycolipids, which were purified by Iatrobeads column and preparative thin-layer chromatographies. One of the glycolipids was found to be globoside, and the other had a globotriaosyl structure with an additional terminal Gal alpha 1-3 residue. Its structure was determined by fast atom bombardment mass spectrometry, two-dimensional proton nuclear magnetic resonance spectrometry (2D NMR), permethylation study, sequential degradation with exoglycosidase, and mild acid hydrolysis to be Gal(alpha 1-3)Gal(alpha 1-4)Gal(beta 1-4)Glc(beta 1-1')Cer.     

Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: a 600-MHz NMR study

Specific lectin-carbohydrate interactions between certain oral streptococci and actinomyces contribute to the microbial colonization of teeth. The receptor molecules of Streptococcus oralis, 34, ATCC 10557, and Streptococcus mitis J22 for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii are antigenically distinct polysaccharides, each formed by a different phosphodiester-linked oligosaccharide repeating unit. These streptococci all coaggregated strongly with both A. viscosus and A. naesludii strains, whereas S. oralis C104 interacted preferentially with certain strains of the latter species. Receptor polysaccharide was isolated from S. oralis C104 cells and was shown to contain galactose, N-acetylgalactosamine, ribitol, and phosphate with molar ratios of 4:1:1:1. The 1H NMR spectrum of the polysaccharide shows that it contains a repeating structure. The individual sugars in the repeating unit were identified by 1H coupling constants observed in E-COSY and DQF-COSY spectra. NMR methods included complete resonance assignments (1H and 13C) by various homonuclear and heteronuclear correlation experiments that utilize scalar couplings. Sequence and linkage assignments were obtained from the heteronuclear multiple-bond correlation (HMBC) spectrum. This analysis shows that the receptor polysaccharide of S. oralis C104 is a ribitol teichoic acid polymer composed of a linear hexasaccharide repeating unit containing two residues each of galactopyranose and galactofuranose and a residue each of GalNAc and ribitol joined end to end by phosphodiester linkages with the following structure. [----6)Galf(beta 1----3)Galp(beta 1----6)Galf(beta 1----6)GalpNAc(beta 1----3) Galp(alpha 1----1)ribitol(5----PO4-]n     

Structural characterization of lactotetraosylceramide, a novel glycosphingolipid isolated from human meconium.

In the course of work on a systematic structural mapping of nonacid glycosphingolipids of human meconia, special attention was given to a major component preliminarily identified as an isomer of neolactotetraosylceramide (paragloboside). This component was isolated in its pure form from meconium of a blood group O individual and subjected to detailed structural analyses, using mass spectrometry and proton NMR spectroscopy on intact permethylated and permethylated-reduced (LiAlH4) derivatives, and gas liquid chromatography on degradational products of native, permethylated, and permethylated-reduced derivatives. The isolated compound was conclusively shown to have the structure Galp beta 1 yields 3GlcNAcp beta 1 yields 3Galp beta 1 yields 4Glcp beta 1 yields 1Cer, and is thus identified as lactotetraosylceramide. The major fatty acids were 2-hydroxy fatty acids with 16 and 20 to 24 carbon atoms, and the bases were sphingosine and phytosphingosine. This glycolipid, although not isolated and structurally characterized before, has long been thought of as a precursor substance of the Lewis active glycolipids and of ABH-active glycolipids with a type 1 saccharide chain.     

New structural features of the polysaccharide from gum ghatti (Anogeissus latifola)

Methylation and 13C NMR analyses were carried out on the high-arabinose, acidic heteropolysaccharide of gum ghatti and the products obtained on three successive, controlled Smith degradations. The side chains contained mainly 2-O- and 3-O-substituted Araf units. Of these the second degradation eliminated remaining alpha-Araf units, and their beta anomers became evident. The proportion of Galp units gradually increased in the form of nonreducing end- and Galp units, although 3,6-di-O- and 3,4,6-tri-O-substituted Galp units diminished. After three degradations groups with consecutive 3-O-substituted beta-Galp units were formed. The proportion of periodate-resistant 3-O- and 2,3-di-O-substituted Manp units was maintained. As a guide to side-chain structures in the polysaccharide, seven of the 10 free reducing oligosaccharide fractions (PC) present in the gum were isolated and examined (NMR, ESIMS, and sometimes methylation analysis). Characterized are alpha-Araf-(1 --> 2)-Ara and three Ara-containing oligosaccharide fractions containing 2-O- and 3-O-substituted units. These gave respectively, ESIMS molecular ions arising from Ara(2), beta-Araf oligosaccharides with four units, beta-Araf oligosaccharides with seven units, and Hex(2)-Ara(4). Alpha-Rhap-(1 --> 4)-GlcA, alpha-Rhap-(1 --> 4)-beta-GlcpA-(1 --> 6)-Gal, and alpha-Rhap-(1 --> 4)-beta-GlcpA-(1 --> 6)-beta-Galp-(1 --> 6)-Gal represented other side chains.     

Comparative structural and molecular characterization of Streptococcus pneumoniae capsular polysaccharide serogroup 10

Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had β1-6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1-2 or wcrF-dependent α1-4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had β1-3-linked Galf branches linked to GalNAc, only CPS10A had additional β1-6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The β1-3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent β1-6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.     

Some biomolecules and a partially O-acetylated exo-galactomannan containing β-Galf units from pathogenic Exophiala jeanselmei, having a pronounced immunogenic response

The pathogenic fungus Exophiala jeanselmei (Ej4) was grown in submerged MM medium, glucose being consumed after six days with maximum biomass and EPS production. Cells were extracted with CHCl3-MeOH (2:1, v/v) yielding a product containing 10% lipid, with high levels of unsaturated C(18:1) (43.6%) and C(18:2) (21.0%), 2D-TLC showed the presence of PE (17.7%), PS (11.6%), PC (35.8%), PI (1.2%) and lyso-phospholipids, LPE (10.7%), LPC (2.0%), PA (10.4%), cardiolipin (10.5%) and glucosyl-ceramide. Analysis of EPS-1 (120 kDa) showed a galactomanan, containing a main chain of Manp-(1→2) (24.2%), substituted by side chains containing terminal Galf (16.8%) and Manp (3.5%) and acetyl groups attached at O-6 of terminal Galf. An immune response against antigens was obtained using Balb/C mice. Anti-EPS-1 antibodies recognized purified fraction containing cellular walls very titer and higher than 1:20,000 for EPS. The studied biomolecules showed biotechnological potential and point to important perspectives in diagnosis of fungi and immunomodulatory products.