The neutral glyceroglucolipids of alveolar lavage from rabbit.

Three individual glycolipids have been isolated from the neutral lipid fraction of rabbit alveolar lavage. All three glycolipids contained glucose, glyceryl monoethers and fatty acids, and differed from each other primarily with respect to the number of glucose residues. The structures of these glycolipids were identified by mild alkaline methanolysis, oxidation with periodate and CrO3, and methylation studies, as: Glc(alpha 1 leads to 3)-1,(3)-O-alkyl-2-O-acylglycerol, Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 lead to 6)Glc(alpha 1 leads to 3)-1,(3)-O-alkyl-2-O-acyglycerol, and Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 3)-l,(3)-O-alkyl-2-O-acylglycerol.     

The monoclonal antibody directed to difucosylated type 2 chain (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc; Y Determinant)

One of the monoclonal (AH-6) antibodies prepared by hybridoma technique against human gastric cancer cell line MKN74 was found to react with a series of glycolipids having the Y determinant (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc). The structure of one such glycolipid isolated from human colonic cancer and from dog intestine was identified as lactodifucohexaosyl-ceramide (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide; IV3,III3Fuc2nLc4Cer). The hapten glycolipid did not react with monoclonal antibodies directed to Lea, Leb, and X-hapten structures, and the AH-6 antibody did not react with the X-hapten ceramide pentasaccharide (Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), H1 glycolipid (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), nor with glycolipids having the Leb (Fuc alpha 1 leads to 2Gal beta 1 leads to 3[Fuc alpha 1 leads 4]GlcNAc beta 1 leads to R) determinant. The antibody reacted with blood group O erythrocytes, but not with A erythrocytes. Immunostaining of thin layer chromatography with the monoclonal antibody AH-6 indicated that a series of glycolipids with the Y determinant is present in tumors and in O erythrocytes.     

Isolation and characterization of a sulfated glyceroglucolipid from alveolar lavage of rabbit.

A major acidic glycolipid of the rabbit alveolar lavage has been isolated and characterized. The isolation procedure involved extraction of lipids, column fractionation on DEAE-Sephadex and silicic acid, and thin-layer chromatography. Chemical analysis of the glycolipid revealed the presence of glucose, fatty acids, glycerol mono-ethers and sulfate. By partial acid and alkaline methanolysis, oxidation with periodate and chromium trioxide, and methylation analysis of the native and desulfated compound, the structure of this glycolipid is proposed to be: SO3H-6Glc alpha 1 leads to 6Glc alpha 1 leads to 6Glc alpha 1 leads to 6Glc alpha 1 leads to 3-1,(3)-O-alkyl-2-0-acylglycerol.     

The parasitic trematode Fasciola hepatica exhibits mammalian-type glycolipids as well as Gal(beta1-6)Gal-terminating glycolipids that account for cestode serological cross-reactivity

Neutral glycosphingolipids from sheep-derived Fasciola hepatica liver flukes were isolated and characterized both structurally and serologically. After HPLC fractionation, glycolipids were analyzed by linkage analysis, enzymatic cleavage, and MALDI-TOF as well as electrospray ionization mass spectrometry. Obtained results revealed the presence of two types of neutral glycolipids. The first group represented mammalian-type species comprising globo- and isoglobotriaosylceramides (Gal(alpha1-4)Gal(beta1-4)Glc(1-1)ceramide and Gal(alpha1-3)Gal(beta1-4)Glc(1-1)ceramide, respectively) as well as Forssman antigen (GalNAc(alpha1-3)GalNAc(beta1-3/4)Gal(alpha1-4/3)Gal(beta1-4)Glc(1-1)ceramide). Applying Helix pomatia agglutinin, recognizing terminal alpha-linked GalNAc, to cryosections of adult flukes, the latter glycolipid could be localized to the F. hepatica gut. As Forssman antigen from the parasite and sheep host led to identical MALDI-TOF MS profiles, this glycolipid might be acquired from the definitive host. As a second group, highly antigenic glycolipids were structurally characterized as Gal(beta1-6)Gal(beta1-4)Glc(1-1)ceramide, Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide and Gal(beta1-6)Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide, the latter two structures of which exhibited both isoglobo- or globo-series core structures. Terminal Gal(beta1-6)Gal1-motifs have previously been shown to represent antigenic epitopes of neogala-series glycosphingolipids from tape worms. Using human Echinococcus granulosus infection sera, Gal(beta1-6)Gal-terminating glycolipids could be allocated to the gut in adult liver fluke cryosections. Corresponding neogala-reactive antibodies in F. hepatica infection serum were detected by their binding to E. granulosus and Taenia crassiceps neogala-glycosphingolipids. These antibodies might contribute to the known serological cross-reactivity between F. hepatica and parasitic cestode infections.     

Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases

Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.     

Regulation of hydroxydocosanoic acid sophoroside production in Candida bogoriensis by the levels of glucose and yeast extract in the growth medium.

Cells of Candida bogoriensis produce as a major extracellular lipid 13-[(2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]docosanoic acid 6',6'-diacetate (Ac2Glc2HDA), the diacetylated sophoroside of 13-hydroxydocosanoic acid (HDA), along with mono- and unacetylated derivatives. The HDA glycolipid production is greater than 2 g/liter when cells are grown on a "standard" medium of 3% glucose and 0.15% yeast extract. Either lowering the glucose concentration (0.5 to 2.0% glucose, at 0.2% yeast extract) or raising the yeast extract concentration (2 to 4% yeast extract at 3% glucose) greatly decreased the yield of this glycolipid, as well as its rate of synthesis measured by [14C]acetate incorporation. Total HDA production was also depressed on the low glucose medium, as was the activity of UDP-glucose:HDA glucosyltransferase, the first enzyme involved in the synthesis of Ac2Glc2HDA from HDA. Levels of acetyl-CoA:Glc2HDA acetyltransferase were not decreased by growth on a low glucose medium, however, even under conditions in which glycolipid production was less than 4% of that found in the standard medium. Low levels of the HDA glycolipids were monitored by high pressure liquid chromatography of their p-bromophenacyl esters, formed by the action of alpha,beta-dibromoacetophenone on the sodium salt of the lipid in the presence of a crown reagent catalyst. This regulation of extracellular Ac2Glc2HDA production by the nutrient composition of the growth medium may represent an important property in the adaptation of C. bogoriensis to its natural environment, the phyllosphere.     

Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine

Escherichia coli K12, which possess the K99 plasmid and synthesize K99 fimbriae (E. coli K99), cause severe neonatal diarrhea in piglets, calves, and lambs but not in humans. The organism binds specifically and with high affinity to only two glycolipids in piglet intestinal mucosa as demonstrated by overlaying glycolipid chromatograms with 125I-labeled bacteria. These glycolipids, which are N-glycolyl-GM3 (NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) and N-glycolylsialoparagloboside (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), occur at about 13 and 0.3 micrograms per gram wet weight of mucosa, respectively. E. coli K99 grown at 18 degrees C, a temperature at which the K99 fimbriae are not expressed, do not bind to these glycolipids. Of the standard glycolipids tested in solid phase binding assays, E. coli K99 binds with highest affinity to N-glycolylsialoparagloboside, with less affinity to N-glycolyl-GM3, and with very low affinity to N-acetylsialoparagloboside. The bacteria do not bind to GM3 (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer), GM2 (GalNAc beta 1-4[Neu-Ac alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), GM1 (Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), or several other N-acetylsialic acid-containing gangliosides and neutral glycolipids at the levels tested. N-Glycolylsialyl residues are found in the glycoproteins and glycolipids of piglets, calves, and lambs but not in the glycoproteins and glycolipids of humans. Possibly this distribution of sialyl derivatives explains the host range of infection by the organism.     

A new extended globoglycolipid carrying the stage specific embryonic antigen-1 (SSEA-1) determinant in mouse kidney

Two neutral glycolipids carrying the stage specific embryonic antigen-1 (SSEA-1) and SSEA-3 determinants, respectively, were purified from mouse kidney by a combination of column chromatographies and droplet counter-current chromatography. The structures of the glycolipids (GL-X and GL-Y) were determined by means of GLC, 1H-NMR spectroscopy, negative-ion fast atom bombardment mass spectrometry, a methylation study, and sequential degradation. GL-X was demonstrated to be galactosyl beta 1-3globotetraosylceramide, the structure of which had already been characterized to be that of SSEA-3 by Kannagi et al. [1983) J. Biol. Chem. 258, 8934-8942). GL-Y was a new glycolipid containing fucose, galactose, glucose, N-acetylgalactosamine, and N-acetylglucosamine in a molar ratio of 1:4:1:1:1. The methylation study results indicated that it contained 3 mol of terminal sugars composed of 2 mol of galactose and 1 mol of fucose with two branching points at N-acetylgalactosamine and N-acetylglucosamine. From the data obtained by 1H-NMR spectroscopy, mass spectrometry, and a binding assay using an anti-SSEA-1 monoclonal antibody (PM81) cloned by Ball et al. [1983) J. Immunol. 130, 2937-2941), we propose the structure of GL-Y to be Gal beta 1-4GlcNAc beta 1-6GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-ceramide. (sequence; see text) Fuc alpha 1 Gal beta 1 This is the first report on the isolation and characterization of a glycolipid carrying the SSEA-1 determinant on its globo-core structure.     

Glycolipid transfer protein from pig brain transfers glycolipids with beta-linked sugars but not with alpha-linked sugars at the sugar-lipid linkage

The glycolipid transfer protein purified from pig brain facilitates the transfer of various glycosphingolipids and glyceroglycolipids (Yamada, K., Abe, A. and Sasaki, T. (1985) J. Biol. Chem. 260, 4615-4621). In this paper, the transfer of Man beta 1----4Glc beta 1-Cer and Man alpha 1----4Man beta 1-Cer isolated from a bivalve, Corbicula japonica, the transfer of 3-[Glc alpha 1-]-sn-1,2-diacylglycerol and 3-[Glc alpha 1----2Glc alpha 1-]-sn-1,2-diacylglycerol prepared from Streptococcus lactis, and the transfer of 3-[Glc beta 1-]-rac-1,2-dipalmitylglycerol have been investigated. The transfer of these lipids from liposomes to mitochondria was assayed by the decrease of these lipids in the donor liposomes. These lipids were determined by chromatographic isolation of the lipids, acid hydrolysis of the isolated lipids, and subsequent determination of glucose in the hydrolysate. The glycolipid transfer protein facilitated the transfer of ManGlcCer and ManManGlcCer. The transfer protein did not facilitate the transfer of Glc alpha-diacylglycerol or Glc alpha Glc alpha-diacylglycerol. However, the transfer of Glc beta-dipalmitylglycerol was facilitated by the protein. These results strongly suggest that the glycolipid transfer protein has the specificity to the presence of beta-linked glucose or galactose directly linked to either ceramide or diacylglycerol.     

Accumulation of unique globo-series glycolipids in PC 12h pheochromocytoma cells

In a previous paper, we reported the presence of a unique globo-series glycolipid as one of the major neutral glycolipid: Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1' Cer, in the subcloned PC 12h pheochromocytoma cells (Ariga, T., Yu, R. K., Scarsdale, J. N., Suzuki, M., Kuroda, Y., Kitagawa, H., and Miyatake, T. (1988) Biochemistry 27, 5335-5340). Recently we found that the subcloned PC 12h cells accumulated other unusual neutral glycolipids. In order to characterize these glycolipids, PC 12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated four minor neutral glycolipids, which were purified by droplet counter-current, Iatrobeads column, and preparative thin-layer chromatographies. These glycolipid structures were determined by fast atom bombardment-mass spectrometry, proton nuclear magnetic resonance spectroscopy, permethylation study, and sequential degradation with various exoglycosidases to be as follows: A, Fuc alpha 1-2Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; B, GalNAc beta 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; C, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; and D, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer. Glycolipids A and B were tentatively characterized in normal rat small intestine (Breimer, M. E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 557-568; Angstrom, J., Breimer, M. E., Falk, K.-E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 682-688). Glycolipids C and D have not been reported in the literature.     

Characterization of two glucuronic acid-containing glycosphingolipids in larvae of the green-bottle fly, Lucilia caesar

Two glucuronic acid-containing glycosphingolipids were purified from larvae of the green-bottle fly, Lucilia caesar by DEAE-Sephadex and Iatrobeads column chromatography. Structures of these acidic glycolipids, glycolipids X and Y, were elucidated by means of sugar analysis, permethylation, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and NMR studies. Glycolipid X was determined to have the following structure: GlcA beta 1-3Gal beta 1-3GalNAc alpha 1-4 GalNAc beta 1-4 GlcNAc beta 1-3Man beta 1-4Glc beta 1-1 ceramide. The other acidic glycolipid, glycolipid Y contains a phosphoethanolamine residue linked through the 6-hydroxy group of the N-acetyl-glucosamine unit of glycolipid X. The ceramide moieties were composed of saturated fatty acids (16:0-22:0) and tetradeca- and hexadeca-4-sphingenines. Based on the structural similarity of the ceramide moieties it appears likely that glycolipid X is an intermediate from which glycolipid Y is synthesized by addition of a phosphoethanolamine residue.     

Biosynthesis of mammary glycoproteins. Partial characterization of the sequence for the assembly of lipid-linked saccharides

Incubation of a membrane preparation from the lactating bovine mammary gland with UDP-[3H]GlcNAc, GDP-[14C]Man, and UDP-[3H]Glc results in the biosynthesis of 15 lipid-linked saccharides that differ from one another by a monosaccharide unit. Pulse and chase kinetics indicate that these glycolipids are related to one another as precursor products for the biosynthesis of asparagine-linked glycoproteins of this tissue. [Man-14C]- and [Man-14C, GlcNAc-3H]saccharides were prepared from corresponding glycolipids by mild acid hydrolysis. Following extensive purification by paper and gel filtration chromatography, structural characterization was conducted on tri-, tetra-, penta-, and undecasaccharides via size determination on calibrated columns of Bio-Gel P-2 and P-4, compositional analysis, exo- and endoglycosidase digestions, methylation, Smith degradation, and acetolysis. These structures were identified as: Man beta 1 leads to 4(3)GlcNAc beta 1 leads to 4(3)Glc-NAc, Man alpha 1 leads to 3Man beta 1 leads to 4(3)GlcNAc beta 1 leads to 4(3)GlcNAc, Man alpha 1 leads to 3(Man alpha 1 leads to 6)Man beta 1 leads to 4(3)Glc NAc beta 1 leads to 4(3)Glc-NAc, and Man alpha 1 leads to 2 Man alpha 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 2Man alpha 1 leads to 6[Man alpha 1 leads to 2Man alpha 1 leads to 3]Man alpha 1 leads to 6)Man beta 1 leads to 4(3)GlcNAc beta 1 leads to 4(3)GlcNAc.     

Pseudomonas aeruginosa and Pseudomonas cepacia isolated from cystic fibrosis patients bind specifically to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2)

Pseudomonas aeruginosa infection in the lungs is a leading cause of death of patients with cystic fibrosis, yet a specific receptor that mediates adhesion of the bacteria to host tissue has not been identified. To examine the possible role of carbohydrates for bacterial adhesion, two species of Pseudomonas isolated from patients with cystic fibrosis were studied for binding to glycolipids. P. aeruginosa and P. cepacia labeled with 125I were layered on thin-layer chromatograms of separated glycolipids and bound bacteria were detected by autoradiography. Both isolates bound specifically to asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) and asialo GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) but not to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), globoside (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer), paragloboside (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), or several other glycolipids that were tested. Asialo GM1 and asialo GM2 bound the bacteria equally well, exhibiting similar binding curves in solid-phase binding assays with a detection limit of 200 ng of either glycolipid. Both isolates also did not bind to GM1, GM2, or GDla suggesting that substitution of the glycolipids with sialosyl residues prevents binding. As the Pseudomonas do not bind to lactosylceramide, the beta-N-acetylgalactosamine residue, positioned internally in asialo GM1 and terminally in asialo GM2, is probably required for binding. beta-N-Acetylgalactosamine itself, however, is not sufficient as the bacteria do not bind to globoside or to the Forssman glycolipid. These data suggest that P. aeruginosa and P. cepacia recognize at least terminal or internal GalNAc beta 1-4Gal sequences in glycolipids which may be receptors for these pathogenic bacteria.     

Characterization of a major neutral glycolipid in PC12 cells as III3Gal alpha-globotriaosylceramide by the method for determining glycosphingolipid saccharide sequence with endoglycoceramidase

Neutral glycolipids in PC12 cells were examined. A major neutral glycosphingolipid, isolated from a chloroform/methanol extract of the cells, was found to contain only galactose and glucose at a ratio of 3:1 and identified as ceramide tetrahexoside by fast atom bombardment (FAB) mass spectrometry. Its saccharide sequence was determined by a new method developed here using endoglycoceramidase (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). The glycosphingolipid was digested with endoglycoceramidase to produce oligosaccharide which was subsequently pyridylaminated. The fluorescence-labeled oligosaccharide was digested with a series of specific exoglycosidases and fractionated by high performance liquid chromatography. The 2-aminopyridyl oligosaccharide was hydrolyzed by alpha-galactosidase to give a 2-aminopyridyl oligosaccharide which was identified as 2-aminopyridyl lactose by high performance liquid chromatography, indicating the glycolipid structure to be Gal alpha Gal alpha Gal beta GlcCer. Ceramide trihexoside obtained by limited digestion of the intact glycolipid was clearly identical with ceramide trihexoside obtained from human erythrocytes, according to NMR spectroscopy and methylation analysis. From these and other data on the intact glycolipid, obtained by methylation analysis and NMR spectroscopy, its structure was confirmed as Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer, III3-Gal alpha-globotriaosylceramide. This is the first report indicating the presence of this glycosphingolipid in PC12 cells.     

Structural studies on glycolipid of shellfish. III. Novel glycolipids from Turbo cornutus

Five kinds of sphingoglycolipids were isolated from Turbo cornutus. Four of them were a series of novel glycolipids consisting only of galactose. The structures of these glycolipids were studied by methylation analysis, periodate oxidation, enzymatic degradation, and proton magnetic resonance spectroscopy. Three glycolipids were characterized as galactosyl(beta 1 leads to 1)ceramide, galactosyl(beta 1 leads to 6)galactosyl(beta 1 leads to 1)ceramide, and galactosyl(beta 1 leads to 6)galactosyl(beta 1 leads to 6)galactosyl(beta 1 leads to 1)ceramide. Data indicating that the 4th glycolipid might be the tetragalactosyl derivative of this series were obtained. The carbohydrate moiety of the 5th glycolipid, in contrast, was composed of fucose, galactose, glucose and N-acetylglycosamine in a molar ratio of 1 : 2 : 1 : 1.     

Location of acyl groups of trehalose-containing lipooligosaccharides of mycobacteria

A variant of a Mycobacterium sp. originating in a patient with Crohn's disease, but not necessarily implicated in the disease, provided a simple version of a newer class of species-specific surface glycolipids, the trehalose-containing lipooligosaccharides. A combination of high-resolution 1H nuclear magnetic resonance, methylation, ethylation, and absolute configurational analysis established the structure of the oligosaccharide unit as beta-D-Glcp(1----3)-alpha-L-Rhap(1----3)-alpha-D-Glcp(1----1)-alph a-D-Glcp (where Glc is glucose, Rha is rhamnose, and p is pyranosyl), and gas chromatography-electron impact mass spectrometry allowed identification of the fatty acyl esters as primarily 2,4-dimethyltetradecanoate. The relative simplicity of the glycolipid combined with the application of a mild methylation procedure and californium-252 plasma desorption mass spectrometry allowed recognition of three such acyl residues on the 3-, 4-, and 6-hydroxyl positions of the terminal glucosyl residue of the trehalose unit. Thus, the glycolipid is decidedly amphipathic yet is clearly not membranous. This observation leads to speculation about the role of these novel lipooligosaccharides in contributing to the outer segment of the hydrophobic barrier of the cell wall of certain mycobacteria.     

Glycolipid antigens of human polymorphonuclear neutrophils and the inducible HL-60 myeloid leukemia line

Glycolipid and cell surface carbohydrate antigens of human polymorphonuclear neutrophils (PMN) and of HL-60 myeloid leukemia cells were analyzed with a panel of defined, monoclonal anti-carbohydrate antibodies. Antigenicities of intact PMN, HL-60, and retinoic acid-induced HL-60 (r.a.-HL-60) were studied by flow cytofluorometry. These three cell populations displayed quantitative differences, some of which were induction dependent, in their expression of lactosyl, N-acetyllactosaminyl, Y-hapten (Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----R), and sialosyl-X-hapten (SA alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----R) specificities. Structures reactive with antibodies specific for long-chain mono-, and di- or tri- alpha 1----3 fucosylated lacto-series glycolipids were also detected. Glycosphingolipids purified from organic extracts of these cells were analyzed to seek information concerning the chemical basis for these surface antigenic differences, to assess the structural and antigenic diversity of PMN and HL-60 glycolipids, and to quantitate chemically and antigenically prominent glycolipids. Binding of monoclonal antibodies to thin-layer chromatograms demonstrated that each of the specificities on intact cells was carried by one or more distinct glycolipids. The abundance of immunoreactive glycolipids in the extracts paralleled the relative staining intensities of the intact cell populations. Several "cryptic" glycolipid antigens, including alpha 2----6 sialosylated structures enriched five- to 10-fold in PMN extracts, were not detected on intact cells. Lactosylceramide accounted for two-thirds of the approximately 1.5 X 10(9) glycolipid molecules contained in each PMN. The remaining glycolipid antigens appeared to include structurally diverse fucolipids, fucogangliosides, and neutral and sialosylated glycolipids with Gal beta 1----4GlcNAc beta 1----R terminal core structure. The abundance, diversity, and induction-dependent expression of these structures suggest that they may participate in PMN maturation and function.     

Characterization of novel glycolipid antigens with an α-galactose epitope in lactobacilli detected with rabbit anti-Lactobacillus antisera and occurrence of antibodies against them in human sera

Anti-Lactobacillus johnsonii (LJ) antisera generated by immunization of rabbits with LJ reacted with glyceroglycolipids in LJ, i.e. dihexaosyl diacylglycerol (DH-DG), trihexaosyl DG (TH-DG) and tetrahexaosyl DG (TetH-DG), whose reactivities with antisera increased proportionally with longer carbohydrate chains of glycolipids. Structural analyses of glycolipids from LJ revealed that DH-DG was Galα1-2Glcα1-3'DG, and TH-DG and TetH-DG were novel derivatives of it with α-Gal at the non-reducing terminal, i.e. Galα1-6Galα1-2Glcα1-3'DG and Galα1-6Galα1-6Galα1-2Glcα1-3'DG, respectively. DH-DG was commonly present in several lactobacilli examined, but TetH-DG was restricted to LJ, L. intestinalis and L. reuteri, while the TH-DGs from L. casei were Glc1-6Galα1-2Glcα1-3'DG and an esterified derivative of it, Glc1-6Galα1-2Glc(6-fatty acid)α1-3'DG, as reported in the literature. Anti-LJ antisera reacted with TH-DG and esterified TH-DG from L. casei to lesser extents, but not at all with gentibiosyl DG from Staphylococcus epidermidis or kojibiosyl DG from Streptococcus salivalis or sphingoglycolipids containing α-Gal residues. The major molecular species of glycolipids obtained from lactobacilli were 11-octadecenoic and 11,12-methylene-octadecanoic acids-containing ones. Also, human IgM antibodies against TH-DG and TetH-DG from LJ were detected in human sera, with various antibody titres, indicating that an immune reaction to symbiotic lactobacilli occurs against their glycolipid antigens, TH-DG and TetH-DG.     

Studies on the relationship between glycerophosphoglycolipids and lipoteichoic acids. IV. Trigalactosylglycerophospho-acylkojibiosyldiacylglycerol and related compounds from Streptococcus lactis Kiel 42172.

Streptococcus lactis Kiel 42172 contains at least six unusually polar glycerophosphoglycolipids. The predominant one was composed of D-galactose, D-glucose, glycerol, acyl groups and phosphorus in a molar ratio of approx. 3 : 2 : 2 : 3 : 1. By analysis of the breakdown products of HF hydrolysis and Smith-degradation the structure was established to be [Galp (alpha 1 leads to 6)Galp(alpha 1 leads to 3)-sn-glycero(2 comes from 1 alpha Galp)-1-phospho] leads to 6Glcp(alpha 1 leads to 2), acyl leads to Glcp(alpha 1 leads to 3)-acyl2Gro. By HF hydrolysis the other compounds were shown to be in the main also derivatives of GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro but they released as water-soluble glycosides Gal(alpha 1 leads to 2)Gro, Gal(alpha 1 leads to 3)Gro, Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), Gal(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro and Gal(alpha 1 leads to 6)Gal-(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), respectively. In the lipid extract Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro and GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3) acyl2Gro were also observed. This set of compounds is proposed to constitute a biosynthetic series reflecting the individual steps in the synthesis of the lipoteichoic acid of Streptococcus lactis Kiel 42172 which is made up by the same lipid anchor and a non-classical poly(galabiosyl, galactosyl glycerophosphate)-chain (Koch, H.U. and Fischer, W. (1978) Biochemistry 17, 5275--5281).     

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.