Substrate specificity of lipase B from Candida antarctica in the synthesis of arylaliphatic glycolipids

Arylaliphatic glycolipids are known for their pharmaceutical and medicinal properties. We found that a great variety of arylaliphatic esters can be synthesized from non-activated substrates like glucose or the natural occurring drug salicin using lipase B from Candida antarctica (CAL-B). However, esters based on aromatic carboxylic acids or unsaturated arylaliphatic acids, like cinnamic acid and its derivatives, which are known to display anticancer activity, could not be obtained. In this work, we performed computer-aided molecular modeling based on data of our work published recently and syntheses of new glycolipids to understand why some substances are accepted by CAL-B while some are not. For this purpose, we investigated the accessibility of the lipase binding site for the arylaliphatic acyl donors as well as the steric interactions between the aglycons of glucosides and the residues of the alcohol binding pocket in order to elucidate potentials and limitations of CAL-B for the synthesis of aromatic glycolipids.     

Syntheses of the 3- and 4-thio analogues of 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and galactopyranoside

The syntheses of 4-nitrophenyl beta-glycosides of the 3-thio and 4-thio analogues of the two principal 2-acetamido-2-deoxy-hexoses found in living systems, GlcNAc and GalNAc, are described. While synthesis of the 4-thio analogues could be achieved via nucleophilic displacements of sulfonate derivatives with thioacetate, problems with neighbouring group acetamido participation necessitated the use of sulfamidate intermediates for the 3-thio analogues. These 3- and 4-thio analogues are employed in the chemo-enzymatic synthesis of thio-oligosaccharide analogues of structures present in glycosaminoglycans, glycoproteins and glycolipids.     

Syntheses of p-nitrophenyl 3- and 4-thio-β-D-glycopyranosides

Thioglycosides have proved to be useful, enzymatically stable analogs of glycosides for structural and mechanistic studies and their synthesis is considerably simplified through the use of thioglycoligases. As part of an investigation into the use of thioglycosides as potential pharmacological chaperones, and as components of glycoproteins and glycolipids, the syntheses of p-nitrophenyl 3-thio-β-d-galactopyranoside, phenyl 1,4-dithio-β-d-glucopyranoside, p-nitrophenyl 4-thio-β-d-mannopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-4-thio-β-d-mannopyranoside are described.     

Phosphatidylinositol mannosides: synthesis and adjuvant properties of phosphatidylinositol di- and tetramannosides

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of glycolipids with significant immune modulating properties. We present here the syntheses of phosphatidylinositol dimannoside (PIM2, 1) and phosphatidylinositol tetramannoside (PIM4, 2) and evaluate their adjuvant properties in a transgenic mouse model. The key step in the synthetic methodology for the synthesis of 2 relies on the selective glycosylation of diol 3 with mannosyl donor 11. Both synthetic PIMs were effective at enhancing IFN-gamma when given as adjuvants with a model antigen, with PIM2 being the more active. These data suggest that in this assay the PIM core structure is responsible for the observed biological activity.     

Synthesis and inhibitory activity of a di- and a trisaccharide corresponding to an erythrocyte glycolipid responsible for the nor polyagglutination

The polyagglutinable erythrocytes NOR contain unusual neutral glycolipids reactive with anti-NOR antibodies. The disaccharide alpha-D-Galp-(1-->4)-D-GalpNAc and the trisaccharide alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1-->3)-D-Gal corresponding to the non-reducing end of a NOR glycolipid (NOR1) were chemically synthesized. The syntheses were based on a common (1-->4)-beta-D-GalNAc precursor, and utilized benzyl glycoside and benzyl ether functions for persistent blocking of hydroxyls. The alpha-D-Galp-(1-->4)-beta-D-GalpNAc structural element has been found only recently in Nature, and derivatives thereof have not been synthesized before. Both the synthesized oligosaccharides inhibited specifically human anti-NOR antibodies, the trisaccharide being 300 times more active than the disaccharide.     

Glycosphingolipid patterns in primary mouse kidney cultures

Primary kidney cultures from C57BL/6J mice, 6 weeks of age or older, were produced using D-valine medium to select for epithelial cell growth. After allowing the cells to attach and proliferate for 1 week following plating, medium was changed once per week. Cells formed nearly confluent monolayers during the second week of culture. The cultured cells contained all of the glycosphingolipids seen in the adult kidney, analyzed by high performance liquid chromatography as their perbenzoyl derivatives. Glucosylceramide, however, was highly predominant in the cultured cells, whereas dihexosyl- and trihexosylceramides predominate in the intact kidney. Sex differences in glycolipid contents found in the intact kidney were also apparent in these cultured cells: The concentration of neutral glycolipids, in general, was higher in male cells than in those derived from females, and the male-specific glycolipid nonhydroxy fatty acid digalactosylceramide was high in male cells but very low in female cells. Neutral glycosphingolipids were labeled in 2-week-old cultures using [3H]palmitate. The [3H]palmitate was incorporated into all of the glycolipids within 2 hr of labeling. Hence, adult mouse kidney cells in D-valine medium retain their differentiated characteristics for a sufficient period of time to allow investigation of glycolipid syntheses in monolayer cultures of epithelial cells derived from this organ.     

Possible role of macrophage glycolipids as receptors for migration inhibitory factor (MIF).

Guinea pig peritoneal exudate cells incubated with water soluble glycolipids obtained from macrophages show an enhanced response to migration inhibitory factor. Incorporation of these glycolipids into liposomes greatly facilitates their interaction with indicator cells. Enhancement of peritoneal exudate cell responsiveness to migration inhibitory factor was specific for glycolipids from guinea pig macrophages. Glycolipids extracted from guinea pig brain and polymorphonuclear leukocytes as well as several bovine and porcine glycolipids had no effect. Specificity of enhancement was not due merely to a preferential association of macrophage glycolipids with indicator cells. The possible role of macrophage glycolipids as receptors for MIF is discussed.     

Highly sensitive gold nanoparticles biosensor chips modified with a self-assembled bilayer for detection of Con A

In this paper, an improved method for detection of Concanavalin A (Con A) with label-free optical biosensors is reported. 1-Dodecanethiol (DDT) was self-assembled onto gold nanoparticles which were deposited on glass slides, and then glycolipid molecules were inserted into dodecanethiol by physical interactions only. The recognition between Con A and carbohydrate was observed by UV-vis spectrophotometry. The absorption spectrum shifted when Con A was bound to the sugar residues of glycolipids immobilized onto nanogold slides, while almost no spectrum change was observed when another nonspecific protein molecule met the nanogold slides. The self-assembled bilayer on nanogold substrates had very high sensitivity for Con A, the minimum detection concentration of Con A can be down to 0.1 nM. In addition to the ultra sensitivity for investigating carbohydrate-lectin interaction, the self-assembled bilayer structure, is expected to replace many receptors which require time-consuming organic syntheses for the fixation to the transducer. The simplicity and sensitivity of this biosensor architecture once again show the prospect of nanogold application in biosensor.     

A novel procedure for the isolation of glycolipids from Bifidobacterium adolescentis 94 BIM using supercritical carbon dioxide

This study describes a novel isolation procedure for major glycolipids from Bifidobacterium adolescentis 94 BIM. The procedure consists of the use of supercritical carbon dioxide (scCO(2)) with hydro-methanolic solution as co-solvent. The major glycolipids were isolated using the following operating conditions: pressure, 30 MPa, co-solvent concentration, 10% (9:1, methanol/water, v/v), CO(2) flow rate, 5 g/min, extraction time and temperature, 2h and 55 degrees C, respectively. The reference glycolipids sample was prepared by classical organic solvent extraction followed by chromatographic purification. All isolates were characterized by TLC and the major glycolipids additionally by enzyme linked immunosorbent (ELISA). Sixty milligrams of glycolipids with similar immunoreactivity as the reference glycolipids were isolated from 1g of freeze-dried biomass (6% of yield).     

Identification of Schistosoma mansoni glycolipids that share immunogenic carbohydrate epitopes with glycoproteins

The immunoreactivity of sera of infected hosts against glycolipids derived from Schistosoma mansoni eggs, adult male worms, and cercariae was analyzed by immunostaining of glycolipids resolved by high-performance thin-layer chromatography. Eggs contained the greatest number of immunogenic glycolipids and bound the largest proportion of serum antibodies. Virtually all of the immunogenic egg glycolipids were neutrally charged and contained oligosaccharide chains larger in size than five sugar residues. The glycolipids of each developmental stage were shown by use of five monoclonal antibodies to share schistosome-specific carbohydrate epitopes that were also present on glycoproteins. Several of the carbohydrate epitopes were expressed throughout the life cycle, yet the overall structures of the glycolipids were not conserved. Quantitative analyses by solid-phase binding assays indicated that the carbohydrate epitopes were differentially expressed between the glycolipids and glycoproteins of developmental stages. Sera from infected humans and mice both contained very high levels of anti-carbohydrate antibodies that were reactive with the glycolipids, irrespective of the stage or intensity of disease. Mice harboring unisexual infections of either male or female worms also recognized the egg glycolipids in a pattern indistinguishable from that of patently infected mice. A greater proportion of the humoral response against egg antigens in infected humans was directed against protein determinants, as compared with infected mice.     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

Lipid composition and molecular species of sialic acids of a mouse cell line (JLS-V9) and its ouabain-resistant mutant

Some properties of a mouse cell line (JLS-V9) and its ouabain-resistant mutant clone (JLS-V9OR) were compared. Specific activities of (Na+ + K+)-ATPase (EC 3.6.1.3) of the cell homogenate, particulate fraction and the plasma membrane fraction were 1.12, 1.72 and 8.75 mu mol/h per mg protein, respectively, in the parent cell and 0.97, 1.68 and 9.36 mu mol/h per mg protein in the mutant cell. The half-maximal concentration of ouabain for the inhibition of the (Na+ + K+)-ATPase was 3.4 . 10(-5) M in the parent cell and 4.0 . 10(-4) M in the resistant clone. The contents of phospholipid and cholesterol on a basis of protein in the mutant were 135 and 105% of those in the parent cell. The mutant's monohexosylceramide (HexCer), lactosylceramide (LacCer), trihexosylceramide (GbOse3Cer) and sialyllactosylceramide (GM3) were 111, 145, 274 and 114% of those of the parent cell. Sialic acids of GM3, analyzed by GC-MS, were 98% N-acetylneuraminic and 2% N-glycolylneuraminic acids in the parent cell, whilst 69% N-acetylneuraminic and 31% N-glycolylneuraminic acids in the mutant clone. The in vivo syntheses of these glycolipids were confirmed by the incorporation of radioactive galactose. No significant difference in fatty acid composition was observed between the two cell types. Neutral glycolipids contained mainly 24:0, 24:1, 22:0 and 16:0, whilst in GM3 18:0 and 18:1 predominated. These results show that the lipid content per mg protein is elevated in the ouabain-resistant cell compared to the parent cell.     

Glycolipids of fetal, newborn, and adult erythrocytes: glycolipid pattern and structural study of H3-glycolipid from newborn erythrocytes

The glycolipids of blood group type O adult, newborn, and fetal erythrocytes were compared. The total amount of glycolipids was indistinguishable between adult and newborn erythrocytes. However, glycolipids with long and neutral carbohydrates and the H determinant were greatly reduced in newborn cells. On the other hand, the amount of sialylated glycolipids (gangliosides) was significantly higher in newborn cells, suggesting that during erythropoiesis sialyltransferases are more active in fetuses than in adults. The amount of each core structure, lacto-N-tetraosyl, linear lacto-N-hexaosyl, and branched lacto-N-octaosyl, was compared between adult and newborn erythrocytes. It was found that branched lacto-series glycolipids were reduced in newborn cells compared with adult cells. Thus, development from fetal to adult human erythrocytes is associated with an increase of branching and a decrease of sialylation of N-acetyllactosaminyl carbohydrate chains. The study indicates that glycolipids are quantitatively different between adult and newborn or fetus.     

Blood group A-active glycosphingolipids analysis by the combination of TLC-immunostaining assay and TLC/SIMS mass spectrometry

Blood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromatographed by TLC and then blood group A-active glycolipids were detected by TLC-immunostaining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glycolipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glycolipids can be readily analyzed by TLC/SIMS.     

Biosynthesis of unnatural glycolipids possessing diyne moiety in the acyl chain in the green sulfur photosynthetic bacterium Chlorobaculum tepidum grown by supplementation of 10,12-heptadecadiynic acid

Unnatural glycolipids possessing the diyne moiety in their acyl groups were successfully biosynthesized in the green sulfur photosynthetic bacterium Chlorobaculum (Cba.) tepidum by cultivation with supplementation of 10,12-heptadecadiynic acid. Monogalactosyldiacylglycerol (MGDG) and rhamnosylgalactosyldiacylglycerol (RGDG) esterified with one 10,12-heptadecadiynic acid were primarily formed in the cells, and small amounts of glycolipids esterified with the two unnatural fatty acids can also be detected. The relative ratio of these unnatural glycolipids occupied in the total glycolipids was estimated to be 49% based on HPLC analysis using a evaporative light scattering detector. These results indicate that the acyl groups in glycolipids, which play important roles in the formation of extramembranous antenna complexes called chlorosomes, can be modified in vivo by cultivation of green sulfur photosynthetic bacteria with exogenous synthetic fatty acids. Visible absorption and circular dichroism spectra of Cba. tepidum containing the unnatural glycolipids demonstrated the formation of chlorosomes, indicating that the unnatural glycolipids in this study did not interfere with the biogenesis of chlorosomes.     

Neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii

The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59 mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosaccharides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal(beta 1-4)Gal-Cer, Gal(beta 1-4)Glc-Cer, and Man(beta 1-4)Glc-Cer; ceramide trisaccharide, Man(alpha 1-3)Man(beta 1-4)Glc-Cer; ceramide tetrasaccharides, Man(alpha 1-3)[Xyl(beta 1-2)]Man(beta 1-4)Glc-Cer, GlcNAc(beta 1-2)Man(alpha 1-3)Man(beta 1-4)Glc-Cer, Man(alpha 1-3)[Gal(beta 1-2)]Man(beta 1-4)Glc-Cer, and Man(alpha 1-2?)Man(alpha 1-3)Man(beta 1-4)Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C18-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.     

Synthesis, biological studies of linear and branched arabinofuranoside-containing glycolipids and their interaction with surfactant protein A

Oligoarabinofuranoside-containing glycolipids relevant to mycobacterial cell wall components were synthesized in order to understand the functional roles of such glycolipids. A series of linear tetra-, hexa-, octa- and a branched heptasaccharide oligoarabinofuranosides, with 1?→?2 and 1?→?5 α-linkages between the furanoside residues, were synthesized by chemical methods from readily available monomer building blocks. Upon the synthesis of glycolipids, constituted with a double alkyl chain-substituted sn-glycerol core and oligosaccharide fragments, biological studies were performed to identify the effect of synthetic glycolipids on the biofilm formation and sliding motilities of Mycobacterium smegmatis. Synthetic glycolipids and arabinofuranosides displayed an inhibitory effect on the growth profile, but mostly on the biofilm formation and maturation. Similarly, synthetic compounds also influenced the sliding motility of the bacteria. Further, biophysical studies were undertaken, so as to identify the interactions of the glycolipids with a pulmonary surfactant protein, namely surfactant protein A (SP-A), with the aid of the surface plasmon resonance technique. Specificities of each glycolipid interacting with SP-A were thus evaluated. From this study, glycolipids were found to exhibit higher apparent association constants than the corresponding oligosaccharide portion alone, without the double alkyl group-substituted glycerol core.     

Biological activity of polar lipids from bifidobacteria

Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics.     

Fatty acids of glycosphingolipids from pig blood fractions

Four glycolipids have been isolated from three fractions of pig blood. The glycolipids were presumably cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The blood fractions were erythrocytes and plasma high and low density lipoproteins. Fatty acid distributions were determined for each glycolipid as a means to assist in identifying relationships among the several glycolipids. Normal fatty acids predominated in all glycolipids except the globosides from erythrocytes in which the amount of hydroxy acids was slightly greater than the amount of normal acids. Hydroxy acids appeared to be present in all the glycolipids, but the concentration was very low in cerebrosides isolated from high density lipoproteins and erythrocytes, and in diglycosyl ceramide and globoside of the low density lipoproteins. In general, the average fatty acid chain length increased from cerebroside to globoside. This was most apparent in erythrocytes and also greater for normal acids than for hydroxy acids. Fatty acid distributions of erythrocyte glycolipids had sufficient variation to make a metabolic relationship by simple addition of a hexose appear doubtful. While the fatty acid distributions found in plasma lipoproteins were more similar, some means of acyl group selection is probably present for either the synthesis or degradation of these glycolipids.     

Biological Activity of Polar Lipids from Bifidobacteria

Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics.