Characterization by mass spectrometry of blood group A active glycolipids from human and dog small intestins.

Glycolipids with blood group A activity isolated from human and dog small intestine have been characterized by mass spectrometry of intact lipid in methylated and in methylated and reduced (LiAiH4) form. Without degradative studies the glycolipids were conclusively shown to be hexaglycosyleramides with phytosphingosine as the major long-chain base and hydroxypalmitic acid as the major fatty acid. The exact sugar ratio was hexose-hexosamine-deoxyhexose 3:2:1 and the sequence established as hexosamine-[deoxyhexose-]hexose-hexosamine-hexose-hexose-ceramide. Evidence is presented that mass spectrometry can differential between type ) and type 2 saccharide chains.     

Identification of a novel hepraglycosylceramide with two fucose residues and a terminal hexosamine.

A polar fucose-containing glycosphingolipid fraction isolated from dog small intestine has been characterized by mass spectrometry of intact methylated, and methylated and reduced (LiAlH4) glycolipid. The native fraction, which was homogenous on thin-layer chromatography, was shown after methylation to be a mixture of two compounds. One was identified as a hexaglycoslyceramide with the following composition and sequence: fucose-hexose(fucose)-hexosamine-hexose-hexose-ceramide, with a terminal saccharide structure similar to blood group Leb determinants. The second compound was a novel heptaglycosyceramide with the sequence: hexosamine(fucose)-hexose-tfucose)-hexosamine-hexose-hexose-ceramide. This glycolipid was also detected in human small intestine and pancreas. The dog intestinal fraction had phytosphingosine as its major base and contained almost exclusively 2-hydroxy fatty acids (16 : 0--24 : 0). The fraction of human pancreas differed in having spingosine as its major base and normal fatty acids (16 : 0--24 :0) as major acids.     

Forssman glycolipid variants of dog gastric mucosa. Structure of a branched ceramide octasaccharide.

A fucose-containing ceramide octasaccharide exhibiting Forssman antigenic activity, and reacting in human H anti-H and anti-A systems, was isolated from water-soluble glycolipids of dog gastric mucosa. Defucosylation of the glycolipid resulted in the loss of H-activity, but had no effect on its Forssman nor blood-group A antigenic activity. The branched structure of glycolipid was identified by partial acid hydrolysis, sequential degradation with specific glycosidases and comparison of the permethylation products of the native and enzyme-degraded compound. The structure of this glycolipid is proposed to be: formula.     

Polarity of the Forssman glycolipid in MDCK epithelial cells

To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.     

Tissue specificity of glycosphingolipids as expressed in pancreas and small intestine of blood group A and B human individuals

A chemical investigation has been done on blood group active glycosphingolipids of both small intestine and pancreas from two individuals, one blood group A and one blood group B. Total non-acid glycolipid fractions were prepared and the major blood group fucolipids present were purified and structurally characterized by mass spectrometry, proton NMR spectroscopy, and degradation methods. The glycolipid structures identified were a blood group Leb hexaglycosylceramide, a B-hexaglycosylceramide with a type 1 (Gal beta 1 leads to 3GlcNAc) carbohydrate chain, A-hexaglycosylceramides with types 1 and 2 (Gal beta 1 leads to 4GlcNAc) carbohydrate chains, a B-heptaglycosylceramide with a type 1 carbohydrate chain, and A-heptaglycosylceramides with type 1 and 2 carbohydrate chains. In addition several minor glycolipids having more than seven sugar residues were detected by thin-layer chromatography. The small intestine and pancreas had some distinct differences in their expression of the major fucolipids. The small intestine contained only glycolipids based upon type 1 carbohydrate chain while the pancreas had both type 1 and type 2 structures. The intestines contained mainly difucosyl compounds while the pancreas tissues contained both mono- and difucosyl glycolipids. Monofucosylglycolipids based on both types 1 and 2 saccharides were present in one pancreas while the other one contained only monofucosylcomponents based on type 1 chain. The ceramides of the intestinal glycolipids were found to be more hydroxylated (trihydroxy long-chain base, hydroxy fatty acids) compared to the pancreas glycolipids (dihydroxy long-chain base, non-hydroxy fatty acids).     

Polysaccharide and glycolipid composition in Tritrichomonas foetus

1. The polysaccharide and glycolipid composition in Tritrichomonas foetus was studied by paper, thin-layer and gas-liquid chromatographic analysis. 2. The carbohydrate components of the polysaccharide were glucose (47%), galactose (34%) and mannose (19%). N-acetylneuraminic acid was the sialic acid derivative characterized in the flagellate whole cells. 3. The sialic acid density was estimated as 2.7 x 10(7) residues/cell. 4. The long-chain base dihydrosphingosine, the carbohydrates galactose (67%), glucose (21%) and mannose (12%) as well as the fatty acids myristic (48%) and palmitic (52%) acids were characterized as components of the total glycolipids of T. foetus. 5. Total glycolipids were fractionated: a galactocerebroside and a ganglioside were identified.     

Characterization of neutral sphingolipids and gangliosides from chicken liver

The neutral sphingolipids and gangliosides were isolated from 62- and 63-day-old chicken livers and characterized. The total concentration of neutral sphingolipids was 59 nmol/g of liver, and that of gangliosides was 330 nmol/g of liver. The major neutral sphingolipids were free ceramide, galactosylceramide, glucosylceramide, lactosylceramide, galabiosylceramide, and Forssman glycolipid. Galactosylceramide was the most abundant and free ceramide was the second most abundant. The major gangliosides were sialosylgalactosylceramide (GM4) and sialosyllactosylceramide (GM3), each of which contained only N-acetylneuraminic acid as a sialic acid. Sphingosine (d18:1) was a major long-chain base in all the sphingolipids. Considerable amounts of 2-hydroxy fatty acids were present in free ceramide, galactosylceramide, and GM4.     

Monoclonal antibodies detecting different epitopes on the Forssman glycolipid hapten

Two hybridomas, derived by fusing mouse myeloma cells with spleen cells from a rat immunized with mouse mammary tumors, have been shown to produce antibodies that recognize cell surface antigens on mesenchymal cells in a variety of tissues. Evidence presented in this report suggests that these antibodies detect overlapping epitopes on the Forssman glycolipid hapten (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). One antibody (33B12) reacts with the terminal sugar sequence GalNAc alpha 1-3GalNAc and is specific for Forssman. The other antibody (117C9) recognizes the internal sugar sequence GalNAc beta 1-3Gal. The terminal sugar sequence GalNAc beta 1-3Gal in globoside, as well as the internal sugar sequence GalNAc beta 1-4Gal in asialo-GM1, is not recognized as an antigenic determinant by 117C9. Nevertheless, the 117C9 antibody does not react exclusively with the Forssman antigen. In a lipid extract fractionated by Folch partition of mouse mammary tumors, the antibody also detects other glycolipids.     

Glycolipids and fatty acids of two dog kidney cell lines.

Glycolipid and fatty acid compositions were studied in whole cells and plasma membranes from two dog kidney cell lines (Madin-Darby and SV40-transformed cells) grown in monolayer and suspension cultures. Glycolipids, which account for 5% or less of the total lipids in dog kidney cells, were substantially increased in plasma membranes relative to whole cells. Sialoglycolipids more complex than a Tay-Sachs-like ganglioside were not found in any whole-cell or plasma-membrane preparation of this study. Dog kidney cells transformed by SV40 virus contained primarily a less complex sialoglycolipid, haematoside. Neutral glycolipids comprised 26-43% of the total glycolipid content in Madin-Darby preparations, whereas in transformed cells and membranes neutral glycolipids constituted only 1-22% of the total glycolipid content. Ceramide trihexoside was found in Madin-Darby cultures, but not in transformed cultures. The values for short-chain fatty acids from neutral glycolipids and for saturated fatty acids were generally higher than the values for these fatty acids in calf serum.     

Structural and compositional difference in the neutral glycolipids between epithelial and non-epithelial tissue of the mouse small intestine

Five major neutral glycolipids, GL-1-GL-5, were isolated from the the mouse small intestine. Their structures and distribution were determined by permethylation analysis, sequential degradation with exoglycosidases and/or immunohistochemistry. The molar ratio of GL-1, GL-2, GL-3, GL-4 and Gl-5 in the whole small intestine was 1:0.04:0.03:0.42:0.02. The structures of GL-1 and GL-4 present in epithelial cells were reported previously to be glucosyl ceramide and asialo G_M1, respectively (Umesaki, Y., Suzuki, A., Kasama, T., Tohyama, K., Mutai, M. and Yamakawa, T. (1981) J. Biochem. 90, 1731–1738). GL-5, also present in the epithelial cells, was fucosyl asialo G_M1, and fucose was shown to be linked to terminal galactose of asialo G_M1 in the manner of α(1–2) bond. GL-2 and GL-3, present in the residual tissue after scraping the mucosa, were determined to be globoside and Forssman glycolipid, respectively. Both globoside and Forssman glycolipid of the non-epithelial tissue had non-hydroxy fatty acid (C₁₆–C₂₄) in combination with sphingosine (C₁₈) as the ceramide components, in contrast with the ceramide structures of the epithelial glycolipids, which contained α-hydroxy fatty acids in combination with phytosphingosine. Immunohistochemical staining using anti-glycolipid antibodies confirmed the distribution of asialo G_M1 and fucosyl asialo G_M1, and Forssman glycolipid in the epithelial and non-epithelial tissue, respectively.     

Determination of the structure of a novel glycolipid from Thermus aquaticus 15004 and demonstration that hydroxy fatty acids are amide linked to glycolipids in Thermus spp.

The compositions of the major glycolipids (GL-1) of five strains of Thermus aquaticus, the type strain of T. filiformis, T. oshimai SPS-11, and Thermnus sp. strain CG-2 were examined by gas chromatography, gas chromatography-mass spectroscopy, fast atom bombardment-mass spectroscopy, and chemical methods. The results showed that, with the exception of T. aquaticus 15004, the organisms each have a major glycolipid whose structure was established as diglycosyl-(N-acyl)glycosaminyl-glycosyl diacylglycerol. Glucosamine was present in GL-1 of T. oshimai SPS-11 and Thermus sp. strain CG-2, while galactosamine was present in the GL-1 of T. aquaticus and T. filiformis. The novel major glycolipid of T. aquaticus 15004 was identified as galactofuranosyl-(N-acetyl)galactosaminyl-(N-acyl)galactosaminyl-gluc - osyl diacylglycerol. The hydroxy fatty acids found in the T. aquaticus strains and in the type strain of T. filiformis were exclusively amide linked to the galactosamine of the major glycolipid. Ester-linked hydroxy fatty acids were not detected in the diacylglycerol moiety of GL-1 of these organisms. Hydroxy fatty acids were detected neither in the major glycolipid of T. oshimai SPS-11 and Thermnus sp. strain CG-2, in which glucosamine is present, nor in the major phospholipid of any of the strains examined.     

Capping of exogenous Forssman glycolipid on cells

When motile cells are incubated with Forssman glycolipid, the antigen is incorporated into the cells' plasma membranes. If cross-linked by antibody, the patched glycolipids cap. This process is sensitive to those drugs that are known to inhibit capping of protein antigens. The results support a flow mechanism for capping.     

New-found phenolic glycolipids in Mycobacterium bovis BCG. Presence of a diglycosylated glycolipid

A crude phenolic glycolipid extract from Mycobacterium bovis bacille Calmette-Guerin (BCG) was fractionated until homogeneity at the intact level into four phenolic glycolipids called B, B-1, B-2, and B-3 according to their polarity. The apolar one, which is the most abundant was assigned to the well-known mycoside B. The B-2 and B-3 phenolic glycolipids were purified by direct-phase high performance liquid chromatography using a 5 micron Spherisorb column but were only recovered in small amounts (3 mg). A linear gradient of 0-20% methanol in chloroform was used. The B-1, B-2, and B-3 glycolipids were subjected to suitable modern analytical techniques selected for their potential to elucidate the structure at the intact level. Desorption chemical ionization-mass spectrometry allowed the molecular mass of B-3 to be determined as 1652 Da for the major homolog establishing the molecular formula as C103H192O14. Thus, the B-3 polar phenolic glycolipid contained two deoxyhexoses, one molecule of phenolphthiocerol esterified by two molecules of mycocerosic acid. Using two-dimensional 1H NMR (correlated chemical shift and nuclear Overhauser effect spectroscopy) at the intact level the B-3 oligosaccharide structure was determined as an alpha-L-Rhap-(1----3)-2-O-Me-alpha-L-Rhap. This is the first report of a diglycosylated phenolic glycolipid in a nonpathogenic mycobacteria. The disaccharide unit, the antigenic determinant, appears to be characteristic of M. bovis BCG. This polar glycolipid B-3 and the apolar ones, B-1 and B-2, were reactive in enzyme-linked immunosorbent assay against serum from rabbit hyperimmunized with M. bovis BCG.     

A basidiomycetous yeast, Pseudozyma crassa, produces novel diastereomers of conventional mannosylerythritol lipids as glycolipid biosurfactants

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These compounds show not only excellent surface-active properties, but also versatile biochemical actions. During a survey of new MEL producers, we found that a basidiomycetous yeast, Pseudozyma crassa, extracellularly produces three glycolipids. When glucose and oleic acid were used as the carbon source, the total amount of glycolipids reached approximately 4.6 g/L in the culture medium. The structures of these glycolipids were similar to those of well-known MEL-A, -B, and -C, respectively. Very interestingly, in all the present glycolipids, the configuration of the erythritol moiety was entirely opposite to that of conventional MELs. The present glycolipids were identified to have the carbohydrate structure of 4-O-β-d-mannopyranosyl-(2R,3S)-erythritol, stereochemically different from 4-O-β-d-mannopyranosyl-(2S,3R)-erythritol of conventional MELs. Furthermore, these new glycolipids possessed both short-chain acids (C₂ or C₄) and long-chain acids (C₁₄, C₁₆, or C₁₈) on the mannose moiety. The major component of the present glycolipids clearly showed different interfacial and biological properties, compared to conventional MELs comprising two medium-chain acids on the mannose moiety. Accordingly, the novel MEL diastereomers produced by P. crassa should provide us with different glycolipid functions, and facilitate a broad range of applications of MELs.     

Control of plastidic glycolipid synthesis and its relation to chlorophyll formation

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv “Halo”) and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [¹⁴C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [¹⁴C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [¹⁴C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [¹⁴C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [¹⁴C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.     

Neutral glycolipid abnormalities in at-complex mutant mouse embryo

The content of neutral glycolipids was studied in normal and twl/twl mutant mouse embryos at embryonic day 11 (E-11). The twl mutation is part of the T/t complex on chromosome 17 and causes embryonic lethality from defects in the developing neural tube. Previous studies suggested that the mutation could involve a defect in ganglioside biosynthesis. Although the total neutral glycolipid content was similar in the normal and mutant whole embryos (approximately 80 nmol glucose/100 mg dry weight), marked differences were detected for the distribution of specific glycolipids. The content of lactosylceramide, globotriaosylceramide, and globotetraosylceramide was significantly higher in the mutant than in the normal embryos, whereas that of glucosylceramide was significantly reduced. The Forssman glycolipid was slightly elevated. The neutral glycolipid composition was similar in embryonic head and body regions of normal embryos, suggesting that the glycolipid abnormalities observed in the mutants are expressed in most embryonic cells and tissues. These and the previously reported ganglioside abnormalities in the twl/twl mutants could result from an inherited defect in glycolipid biosynthesis.     

Forssman penta- and tetraglycosylceramide are xenoantigens of ostrich kidney and liver.

The heterophile antigens Galalpha1-->3Gal and N-glycolylneuraminic acid are the major obstacle to grafting mammal organs, especially from pig, to man. Lack of expression of these common xenoantigens by birds has raised interest in ostrich as a potential organ donor for xenotransplantation. Glycosphingolipids of ostrich liver and kidney were investigated for their carbohydrate determinants. Both organs were found similar in their glycolipid composition with three major species, mono-, di-, and pentaglycosylceramide. The pentaglycosylceramide was characterized as the Forssman antigen. In both organs, the ceramide portion was highly hydroxylated with prevalence of alpha-hydroxylated fatty acids, C18 phytosphingosine in kidney and C18 sphingosine in liver Forssman glycolipid. These data indicate that hydroxylation of kidney glycosphingolipids, which is found in mammals, has been maintained since the divergence of birds from other vertebrates. Characterization of a minor glycolipid as a Forssman tetraglycosylceramide built on the galabiosylceramide core indicates that the Forssman tetraglycosylceramide also exists in vivo. Its precursors, galactosyl- and galabiosylceramide, were characterized in kidney and liver. The Forssman antigen is the third heterophile antigen against which man raises natural antibodies. Its localization in the vascular endothelium and connective tissue makes ostrich an unpromising organ or cell donor for xenotransplantation to man.     

Binding of pulmonary surfactant protein A to galactosylceramide and asialo-GM2.

The binding of pulmonary surfactant protein A (SP-A) to glycolipids was examined in the present study. The direct binding of SP-A on a thin-layer chromatogram was visualized using 125I-SP-A as a probe. 125I-SP-A bound to galactosylceramide and asialo-GM2, but failed to exhibit significant binding to GM1, GM2, asialo-GM1, sulfatide, and Forssman antigen. The study of 125I-SP-A binding to glycolipids coated onto microtiter wells also revealed that SP-A bound to galactosylceramide and asialo-GM2. SP-A bound to galactosylceramides with non-hydroxy or hydroxy fatty acids, but showed no binding to either glucosylceramide or galactosylsphingosine. Excess native SP-A competed with 125I-SP-A for the binding to asialo-GM2 and galactosylceramide. Specific antibody to rat SP-A inhibited 125I-SP-A binding to glycolipids. In spite of chelation of Ca2+ with EDTA or EGTA, SP-A retained a significant binding to glycolipids. Inclusion of excess monosaccharides in the binding buffer reduced the glycolipid binding of SP-A, but failed to achieve complete abolishment. The oligosaccharide isolated from asialo-GM2 is also effective at reducing 125I-SP-A binding to the solid-phase asialo-GM2. From these data, we conclude that SP-A binds to galactosylceramide and asialo-GM2, and that both saccharide and ceramide moieties in the glycolipid molecule are important for the binding of SP-A to glycolipids.