Elucidation of the phenotypic change on the surface of Had-1 cell, a mutant cell line of mouse FM3A carcinoma cells selected by resistance to Newcastle disease virus infection

Had-1, which was isolated from mouse FM3A carcinoma cells, was a non-permissive mutant cell line to Newcastle disease virus infection. Comparative study of the asparagine-linked sugar chains of the surface glycoproteins of the mutant and its parental cells revealed that galactosylation of the complex-type sugar chains is extensively reduced in the mutant. Assay of galactosyltransferase in the two cell lines, however, showed that the enzymatic activity in Had-1 cells is virtually identical to that in FM3A cells. Somatic cell hybridization analysis indicated that the mutant has the same defect as Chinese hamster ovary cell mutant Lec 8, which is deficient in UDP-galactose transport into Golgi vesicles.     

Isolation and characterization of mouse FM3A cell mutants which are devoid of Newcastle disease virus receptors.

A method was developed to select host cell mutants which did not permit the replication of Newcastle disease virus (NDV), and 14 isolates of NDV-nonpermissive mutants of mouse FM3A cells were obtained. All these isolates were judged to be deficient in NDV receptors, since their ability to adsorb 3H-labeled NDV virions was markedly decreased. They were tested for genetic complementation in pairs by cell fusion and shown to fall into a single recessive complementation group, which was designated as Had-1. Vesicular stomatitis virus was able to replicate in this mutant to produce infectious progeny, but the glycoprotein of the released virion was abnormal in size, suggesting a defective processing of the asparagine-linked carbohydrate chains in the mutant cell. The Had-1 mutant was resistant to wheat germ agglutinin, but sensitive to a Griffonia simplicifolia lectin, GS-II, which recognizes terminal N-acetylglucosamine residues. The altered sensitivity to these plant lectins compared with that of the parental FM3A cells indicates that sialylated sugar chains on the cell surface are almost absent from the Had-1 cells, thereby rendering the cells NDV receptor deficient.     

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 of reference standards to enable single cell metabolomic studies of tetramethylrhodamine-labeled ganglioside GM1

Ganglioside GM1 and its seven potential catabolic products: asialo-GM1, GM2, asialo-GM2, GM3, Lac-Cer, Glc-Cer and Cer, were labeled with tetramethylrhodamine (TMR) to permit ultra-sensitive analysis using laser-induced fluorescence (LIF) detection. The preparation involved acylation of the homogenous C(18)lyso-forms of GM1, Lac-Cer, Glc-Cer and Cer with the N-hydroxysuccinimide ester of a beta-alanine-tethered 6-TMR derivative, followed by conversion of these labeled products using galactosidase, sialidase, and sialyltransferase enzymes. The TMR-glycolipid analogs produced are detectable on TLC down to the 1 ng level by the naked eye. All eight compounds could be separated within 4 min in capillary electrophoresis where they could be detected at the zeptomole (ca. 1000 molecule) level using LIF.     

Carbohydrate analysis of chicken heart glycolipids

Neutral and acidic glycolipids were extracted from chicken hearts. The neutral and acidic compounds were separated by preparative thin-layer chromatography into eight and two fractions, respectively. Total hydrolysis by mineral acid, permethylation analysis, and sequential cleavage with exoglycosidases showed the presence of glycolipids that belong to the globo- and gala-oligosaccharide series, i.e., the monohexosylceramides Glc-Cer and Gal-Cer, the dihexosylceramides Gal beta 1-4Glc-Cer and Gal alpha 1-4Gal-Cer, the tetrahexosylceramides GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal-Cer (III3GalNAc alpha-Ga3Cer) and four subfractions of the Forssman glycolipid GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. With the notable exception of III3GalNAc alpha 1-Ga3Cer, all glycolipids with terminal GalNAc alpha 1-3GalNAc1 reacted on thin-layer chromatograms with a monoclonal anti-Forssman antibody. The major components of the acidic fraction glycolipids were characterized as the lactose-based gangliosides Glac1 (GM3) and Glac2 (GD3).     

Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.     

Glycosphingolipid carriers of carbohydrate antigens of human myeloid cells recognized by monoclonal antibodies

Six monoclonal antibodies with known specificities for the carbohydrate antigens i, X or Y, and seven anti-myeloid antibodies (determinants unknown) selected for their differing reaction patterns with human leucocytes were tested in chromatogram binding assays for reactions with myeloid cell glycolipids derived from normal human granulocytes and chronic myelogenous leukemia cells. Antigenicities were found exclusively on minor glycolipids which were barely or not at all detectable with orcinol-sulphuric acid stain. Among these, a neutral glycosphingolipid bound the anti-i antibody Den and chromatographed as the ceramide octasaccharide, Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer. Several species of neutral glycosphingolipids with six to more than ten monosaccharides were detected which carry the X antigen and others the Y antigen: Gal beta 1----4(Fuc alpha 1----3)GlcNAc and Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc, respectively. In addition, three new types of carbohydrate specificities were detected among the myeloid cell glycolipids. Two were associated with neutral glycolipids: the first, recognised by anti-myeloid antibodies VIM-1 and VIM-10, was expressed on a distinct set of glycolipids with six or more monosaccharides, and the second, recognized by VIM-8, was expressed on glycolipids with more than ten monosaccharides. The third specificity, recognised by the anti-myeloid antibody VIM-2, was expressed on slow migrating sialoglycolipids with backbone structures of the poly-N-acetyllactosamine type that are susceptible to degradation with endo-beta-galactosidase. Thus, we conclude that the i and Y antigens occur among the glycolipids of normal myeloid and chronic myelogenous leukemia cells and that a high proportion of hybridoma antibodies raised against differentiation antigens of myeloid cells are directed at carbohydrate structures.     

Establishment of mutant murine mammary carcinoma FM3A cell strains transformed with the herpes simplex virus type 2 thymidine kinase gene

To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively.     

Inhibitory effects of glycolipids fraction from spinach on mammalian DNA polymerase activity and human cancer cell proliferation

We succeeded in purifying the fraction containing the major glycolipids in monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol (SQDG) from dried vegetables. This glycolipids fraction was an inhibitor of DNA polymerase alpha (pol alpha) in vitro and also the proliferation of human cancer cells. In this study, eight common vegetables were investigated in terms of the glycolipids fraction, the amounts of major glycolipids, mammalian DNA polymerase inhibitory activity and antiproliferative activity toward human cancer cells. Green tea possessed the largest amount of glycolipids overall. Spinach contained the largest amount of SQDG, followed by parsley, green onion, chive, sweet pepper, green tea, carrot and garlic. Spinach had the strongest inhibitory effect on pol alpha activity and human cancer cell proliferation. A significant correlation was found between SQDG content and inhibition of DNA polymerase. Therefore, the inhibition of pol alpha activity by SQDG may lead to cell growth suppression. Of the six subspecies of spinach (Spinacia oleracea) tested, "Anna" had the largest amount of SQDG, strongest inhibitory activity toward DNA polymerase and greatest effect on human cancer cell proliferation. Based on these results, the glycolipids fraction from spinach is potentially a source of food material for a novel anticancer activity.     

Brefeldin A induced inhibition of de novo globo- and neolacto-series glycolipid core chain biosynthesis in human cells. Evidence for an effect on beta 1-->4galactosyltransferase activity.

De novo synthesis of neolacto-series glycolipids has been studied in human cell lines via metabolic labeling of ceramide with [3H]serine. Intense labeling of ceramide mono- and dihexoside glycolipids occurred with labeling of progressively longer chain derivatives with increasing time. Most of the label was recovered in neutral glycolipids with about 5% of the total labeling in the ganglioside fraction. Experiments done using cell treatment with 2.5 micrograms/ml brefeldin A resulted in a stimulation in the total amount of labeling, accumulation of a neutral glycolipid identified as Lc3 due to inhibited transfer of the neolacto-series core chain terminal beta-Gal residue, and a corresponding inhibition of labeling of longer chain neutral glycolipids in all cell lines. Brefeldin A also blocked synthesis of the globo-series precursor, Gb3, longer chain sialylated structures such as IV3NeuAcnLc4, but not de novo GM3 synthesis. Brefeldin A treatment had no effect on cellular beta 1-->3N-acetylglucosaminyl-, beta 1-->4galactosyl-, or alpha 1-->3fucosyltransferase specific activities, nor was it inhibitory in beta 1-->4galactosyltransferase assays in vitro. The results describe brefeldin A-induced blocks in globo- and neolacto-series glycolipid biosynthesis, consistent with differential localization of enzymes in intracellular membranes. In particular, the results suggest that the beta 1-->4galactosyltransferase in these cells is either not redistributed by brefeldin A or is otherwise rendered nonfunctional.     

The occurrence of glycosphingolipids containing mannose in the sea-water bivalve, Meretrix lusoria (Hamaguri)

Total neutral and acidic glycosphingolipids were prepared from whole tissues of the sea-water bivalve, Meretrix lusoria, and the former preparation was further fractionated into subgroups by silicic acid column chromatography. The fractions obtained as mono-(ceramide monosaccharide, CMS), di-(CDS) and triglycosylceramides (CTS) were characterized by thin-layer chromatography, partial hydrolysis with exoglycosidases, methylation studies, CrO3 oxidation, and GLC analysis of the component sugars, fatty acids and long-chain bases. The following structures are proposed: Gal-Cer and Glc-Cer for CMS, Gal(beta 1----4)Glc-Cer and Man(beta 1----4)Glc-Cer (MlOse2Cer) for CDS, Man(alpha 1----3)Man(beta 1----4)Glc-Cer (MlOse3Cer) and Gal(alpha 1----3)Man(beta 1----4)Glc-Cer (II3 alpha Gal-MlOse2Cer) for CTS. To our knowledge II3 alpha Gal-MlOse2Cer has not previously been reported. The fatty acid composition of CMS, CDS, and CTS consisted almost entirely of saturated C16-C24 acids with large amounts of 2-hydroxypalmitic acid and 2-hydroxystearic acid. The long-chain bases consisted of 4-sphingenine and 4,8-sphingadienine. More complex neutral glycolipids than CTS, as well as an acidic glycolipid, were examined by TLC and GLC of the constituent sugars, and an immunochemical technique.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gangliosides and neutral glycosphingolipids of normal tissue and oat cell carcinoma of human lung

Concentration and composition of gangliosides and neutral glycosphingolipids of adult human lung, and lung small cell carcinoma were studied. The structures of the glycolipids were determined by quantitative component determination, enzymic degradation, permethylation and fast atom bombardment mass spectrometry. Adult human lung contained mainly gangliosides with lactosylceramide as the basic core, GM3, GD3 and GT3, and approx. equal proportions (10%) of gangliosides of the gangliotetraosyl- and lactotetraosylceramide series. 18 gangliosides with different carbohydrate moieties were identified: four of them were only found in the tumor tissue. The adult human lung contained 85 nmol (77-120) gangliosides and 140 nmol neutral glycosphingolipids per g wet weight. Globoside was the major neutral glycolipid and there were only minor amounts of glycolipids of the lactotetraose series. In small cell carcinoma tissue the concentration of neutral glycosphingolipids was approximately twice as high than in normal lung tissue, and there was a markedly larger concentration of both lactosylceramide and glycolipids of the lactotetraose series and fucose derivatives of these. The concentration of gangliosides varied between 202 and 415 nmol per g wet weight. Compared to normal lung tissue, the tumor tissue had a lower proportion of GD3, and a higher proportion of complex gangliosides, and they contained five tumor-associated gangliosides: Fuc-GM1, Fuc-GD1b, 3'-LM1, Fuc-3'-LM1 and 6'-nLM1.     

Glycosphingolipids of normal bovine and enzootic bovine leukosis lymph node cells

We analyzed glycosphingolipids from normal lymph node cells of seven cattle and lymph node cells of eight cattle with enzootic bovine leukosis. The neutral glycosphingolipids and gangliosides were analyzed by thin-layer chromatography. Both normal and tumorous lymph node cells had GlcCer, LacCer, and GbOse3Cer as major neutral glycosphingolipids. In the ganglioside fraction, GM3 was the predominant component in both normal and tumorous lymph node cells, and another component, ganglioside Gx fraction, was also prominent in tumorous lymph node cells. The structure of this ganglioside Gx fraction was elucidated by thin-layer chromatography, sugar analysis, neuraminidase digestion, and permethylation studies. This ganglioside Gx fraction was found to be a mixture of four ganglioside species. The structures of individual gangliosides Gx (1 to 4) were characterized as follows. 1: GD3, NeuAc alpha 2-8NeuAc alpha 2-3Gal1-4Glc-Cer. 2: GD3, NeuAc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. 3: GD3, NeuGc alpha 2-8NeuAc alpha 2-3Gal1-Glc-Cer. 4: GD3, NeuGc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. These GD3 species may be formed as a result of the induced synthesis inassociation with malignant transformation.     

Gas chromatography/mass spectrometry analysis of oligosaccharides from neutral glycosphingolipids of murine B cell hybridomas

Monoclonal B cell hybridoma cell lines express glycolipids characteristic of both the myeloma and normal lymphocyte parents. The neutral glycolipids from hybridoma cell lines metabolically radiolabeled with [14C]galactose plus [14C]glucosamine separate by high performance thin layer chromatography into patterns that may reflect differences in glycolipid expression among B cell subsets. Gas chromatography/mass spectrometry of oligosaccharides released by trifluoroacetolysis from neutral glycolipids extracted from 10(9) clonally expanded hybridoma cells reveals the carbohydrate composition of the major glycolipid components detected by thin layer chromatography. Glycolipids differentially expressed among six cell lines analyzed include monohexosylceramide, lactosylceramide, globotriaosylceramide, globoside, asialo-GM2, and 2'fucosyllactosylceramide. The latter compound has not been described previously in cells from the mouse.     

Glycolipid changes in murine myelogenous leukemias: neutral glycolipids as markers for specific populations of leukemias

We have studied the glycolipid composition of six different murine myelogenous leukemias as well as that of T-cell leukemias and normal spleen cells. Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC) and an HPTLC overlay method. Murine myelogenous leukemias were found to contain globo- and ganglio-series neutral glycolipids, e.g., glucosylceramide (Glc-cer), lactosylceramide (Lac-cer), globotriaosylceramide (Gb3), globoside (Gb4), Forssman glycolipid (Gb5), and asialo-GM1 (GA1). Monoblastic leukemia cells contained increased proportions of Gb3, Gb4, Gb5, and GA1. Monocytic and myelomonocytic leukemia cells contained increased proportions of Glc-cer and Lac-cer. Especially, Glc-cer accounted for approximately 60% of the total neutral glycolipids in monocytic leukemia cells. Gb3 was the major neutral glycolipid in reticulum cell neoplasm type A, and it accounted for approximately 75% of the neutral glycolipids. GA1 was the major neutral glycolipid in myeloblastic and granulocytic leukemia cells as well as T-cell leukemias. Especially, granulocytic leukemia cells contained predominantly GA1, and it accounted for approximately 80% of the total neutral glycolipids. The pattern of gangliosides in myelogenous leukemias was more complex when compared with that of the neutral glycolipids; murine myelogenous leukemias contained at least 13 gangliosides, including such major gangliosides as GM1, GM1b containing N-acetyl neuraminic acid and N-glycolyl neuraminic acid, and Ga1NAc-GM1b. Alterations of glycolipid composition in murine myeloid leukemias may be associated with cellular differentiation and maturation, and therefore these characteristic glycolipid species may be regarded as markers for specific populations of leukemia cells.     

Chemical structure of carcinoma ganglioside antigens defined by monoclonal antibody C-50 and some allied gangliosides of human pancreatic adenocarcinoma

A hybridoma, C-50, obtained by fusion of mouse myeloma cells with spleen cells from a mouse immunized with cells from the colorectal carcinoma cell line COLO 205, produced antibodies that detected ganglioside antigen in human adenocarcinomas in many organs. The major ganglioside antigen fraction isolated from liver metastases of a pancreatic adenocarcinoma, behaving as a homogenous band on thin-layer chromatography, consisted of three different gangliosides. One of them, A (25%), had the same carbohydrate structure as the ganglioside antigen defined by monoclonal antibody 19-9, NeuAc alpha 2-3Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3Gal beta 1-4Glc-Cer(Fuc-3'-isoLM1) Magnani, J.L., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, Z., Koprowski, H. and Ginsburg, V. (1982) J. Biol. Chem. 257, 14365-14369). The major ganglioside, B (60%), was the isomeric hexasaccharide ganglioside (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3-Gal beta 1-4Glc-Cer(Fuc-3'-LM1) and the third ganglioside, C, was 6'-LM1, NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer (15%). Ganglioside B, isolated from human kidney, did not react with the C-50 MAb. Based on this result and on studies of COLO 205 cell induced tumours where the ganglioside antigen fraction only consisted of A, it is suggested that the C-50 MAb defines an antigen determinant present in A.     

Involvement of a neutral glycolipid in differential cell adhesion in the Xenopus blastula.

Many different molecular species mediate cell adhesion during embryonic development. These can have either protein or carbohydrate functional groups, which can act in either a homophilic or a heterophilic manner, and often in concert. We report here that a monoclonal antibody, M4B, raised against Xenopus blastomere membranes, inhibits the calcium-dependent adhesion of dissociated blastomeres. M4B maintains its inhibitory effect on adhesion when converted into univalent fragments, and specifically affects calcium-dependent adhesion. The antigen is regulated in both space and time during early development. It is found on cell surfaces throughout the egg to blastula stages, but is more concentrated on cells in the animal and marginal zones of the blastula. It is dramatically downregulated during gastrulation, and becomes largely restricted to gut epithelium by the larval stages. We show also that M4B function is spatially differentiated at the blastula stage, since it inhibits the aggregation of dissociated animal cells to a greater extent than vegetal cells. This membrane antigen may therefore play a role in the differential adhesion observed between different regions of the blastula, and which we presume to underlie the segregation of the primary germ layers during gastrulation. M4B recognizes a complex of plasma membrane glycolipids. Periodate treatment destroys the ability of these glycolipids to react with the antibody, indicating that the epitope resides in the carbohydrate moiety of the glycolipids. Chemical characterization shows that it is a neutral glycolipid, and that the major component is of the glycoglycerolipid, rather than the more common glycosphingolipid class. Blocking experiments with oligosaccharides of defined structure, and antibody crossreactivity show that the M4B antibody does not recognize several known embryonic carbohydrate antigens. These results demonstrate that M4B antibody recognizes a novel group of developmentally regulated glycolipids which function in calcium-dependent cell--cell adhesion in the Xenopus blastula.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Structures of glycosphingolipids isolated from human granulocytes. The presence of a series of linear poly-N-acetyllactosaminylceramide and its significance in glycolipids of whole blood cells

Structures of glycolipids isolated from human granulocytes were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase treatment. All neutral glycolipids, with saccharide residues ranging from 2 to 10, were found to have linear N-acetyllactosaminyl backbones. The majority of neutral glycolipids contain one or two fucosyl residues attached to N-acetylglucosamine residues through the Fuc alpha 1----3 linkage and were reactive with the monoclonal antibody specific to Gal beta 1----4(Fuc alpha 1----3)GlcNAc, the Lex structure. Their general structure can be expressed as follows: (formula; see text) where n = 0-3. Glycolipids containing sialic acid (gangliosides) were also found to have linear N-acetyllactosaminyl backbones with sialic acid joined to this backbone by either alpha 2----3 or alpha 2----6 linkage. The gangliosides have the following general structure: (formula; see text) where n = 0-3. The ceramide was composed of sphingosine with d18:1 as the long-chain base and C16:0 (as a major component) or C24:1 (as a minor component) fatty acid. Analysis of glycolipids isolated from granulocytes, erythrocytes, and whole blood cells revealed that, among the glycolipids prepared from the whole blood cells, dihexaosylceramide, lactoneotetraosylceramide, and the above described linear lactoneo series neutral glycolipids are present in granulocytes but barely present in erythrocytes.     

Heparin inhibits specific glycosyltransferase activities in interleukin 2 activated murine T cells

In order to better understand the role of cell surface glycolipids in T lymphocyte activation, heparin was used to simultaneously modulate the expression of glycolipids and the lytic capacity of lymphocytes activated by interleukin-2. Results presented here show that heparin added at the start of a 3 day culture inhibited the formation of lymphokine activated killer cells by up to 50%. Heparin also has a profound effect on the synthesis of glycolipids during this three day period. Asialo GM1, a useful cell surface marker for subsets of murine cytotoxic cells, is reduced in amount, as are the other two major neutral glycolipids lactosylceramide and asialo GM2. In addition, the synthesis of some gangliosides is affected by heparin treatment. Comparison of the glycosyltrasferase activities of untreated and heparin-treated cells shows that the activities of a 2–3-sialyltransferase and a 1–3 galactosyltransferase are inhibited dramatically, while a third enzyme, N-acetyl-galactosaminyltransferase is unaffected. The two heparin inhibitable enzymes bind to heparin affinity columns but the galactosaminyltransferase does not. These studies suggest that the proper regulation of the activities of specific glycosyltransferases may be important events in lymphocyte activation.